CN117402872A - Cleavage and washing reagent for nucleic acid extraction - Google Patents

Cleavage and washing reagent for nucleic acid extraction Download PDF

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CN117402872A
CN117402872A CN202311503582.0A CN202311503582A CN117402872A CN 117402872 A CN117402872 A CN 117402872A CN 202311503582 A CN202311503582 A CN 202311503582A CN 117402872 A CN117402872 A CN 117402872A
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nucleic acid
polyoxyethylene
sodium
acid
reagent
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万茂坤
盛青松
肖建勇
高鹏
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Wuxi Shenrui Bio Pharmaceuticals Co ltd
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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    • 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

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Abstract

The invention discloses a lysis and washing reagent for nucleic acid extraction, wherein a lysis solution comprises a surfactant, a first buffer compound, a chaotropic compound and a chelating complexing agent, a washing reagent comprises a chaotropic compound, a second buffer compound, a nonionic surfactant and alcohol, the surfactant is preferably sulfate or sulfonate of fatty alcohol, in particular a detergent based on bile acid, such as sodium deoxycholate and sodium cholate, and a detergent based on sarcosine, such as sodium deoxycholate and sodium cholate. The invention provides a novel cracking reagent for extracting nucleic acid, which further improves the capability of cracking biological samples to release nucleic acid; the novel washing reagent is provided, the washing capability of the surface of a solid material is further improved, the purity of the extracted nucleic acid is improved, the inhibition to downstream application is smaller, and the nucleic acid detection sensitivity can be effectively improved.

Description

Cleavage and washing reagent for nucleic acid extraction
Technical Field
The invention relates to the field of nucleic acid extraction, in particular to a cracking and washing reagent for nucleic acid extraction.
Background
The cleavage and washing reagent is a reagent for nucleic acid extraction, and high-quality nucleic acid extraction is a precondition of many technologies in the field of molecular diagnosis, such as gene library construction, PCR amplification and blotting analysis, and the extraction of high-quality nucleic acid from a biological sample containing cells must be performed as far as possible to separate the nucleic acid from impurities such as proteins, lipids, polysaccharides and nucleases, which might interfere with the detection of downstream nucleic acids, especially the activities of DNA polymerase, reverse transcriptase, ligase and restriction enzymes, and along with the development of technology, the requirements of the process for manufacturing the cleavage and washing reagent are also increasing.
The prior art has developed a variety of methods for extracting nucleic acids from a biological sample containing cells, which are based mainly on two extraction principles:
the first principle is to add a chaotropic agent and an organic solvent (usually phenol and/or chloroform) to a biological sample, which together destroy the cell membrane and the nuclear membrane, release the nucleic acid into the aqueous phase, and impurities, such as proteins, lipids, to be removed remain in the organic phase for the purpose of isolating the nucleic acid. However, the organic solvents commonly used for nucleic acid extraction (typically phenol and/or chloroform) are toxic to humans.
In view of the drawbacks of the first principle, the prior art has invented a safer and more efficient method, the principle of which is that in a certain solution environment, nucleic acids can be adsorbed by solid materials, such as silica. In a solution containing chaotropic agents and/or alcohols, nucleic acids are contacted and bound to the solid material, while impurities such as proteins, lipids, etc. remain in the solution, after which the solid material is washed one or more times to remove impurities other than nucleic acids, and finally the nucleic acids are eluted from the solid material by means of a suitable solution environment.
In the prior art, silica materials have been fabricated in a variety of forms that facilitate extraction of nucleic acids, most commonly spin columns and magnetic beads. One or more layers of glass fiber membranes are fixed in the center of the centrifugal column, and the magnetic beads are magnetic particles coated with silicon dioxide, and the size of the magnetic particles is in the nanometer and micrometer level. The matched solution comprises a cracking reagent, a binding reagent, a washing reagent and an eluting reagent. Although various commercial kits have been developed based on centrifugal columns and magnetic beads, the lysis reagent still has the problem of incomplete lysis when processing high cell content biological samples such as blood, tissues, semen, cultured bacteria, incompletely solubilized proteins and cell fragments are crosslinked with nucleic acids, co-adsorbed onto solid materials, resulting in clogging of glass fiber membranes in centrifugal columns, or agglomeration of magnetic bead particles, and in the subsequent washing process, washing reagent is difficult to sufficiently wash glass fiber membranes or magnetic bead surfaces, eventually resulting in impure extracted nucleic acids affecting downstream detection, for which we propose a lysis and washing reagent for nucleic acid extraction.
Disclosure of Invention
The technical problems to be solved are as follows: aiming at the defects of the prior art, the invention provides a cracking and washing reagent for extracting nucleic acid, and provides a novel cracking reagent, which further improves the capability of cracking biological samples to release nucleic acid; the novel washing reagent is provided, the washing capability of the surface of a solid material is further improved, the purity of the extracted nucleic acid is improved, the downstream application inhibition is smaller, the nucleic acid detection sensitivity can be effectively improved, and the problems in the background technology can be effectively solved.
The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a lysis and washing reagent for nucleic acid extraction, the lysis solution comprising a surfactant, a first buffer compound, a chaotropic compound and a chelating complexing agent, and the washing reagent comprising a chaotropic compound, a second buffer compound, a nonionic surfactant and an alcohol.
As a preferred embodiment of the present application, the surfactant is preferably a sulfate or sulfonate of a fatty alcohol, in particular a bile acid based detergent such as sodium deoxycholate, sodium cholate, a sarcosine based detergent such as sarcosyl sarcosine, N-lauroyl sarcosine, a sulfate or sulfonate of a fatty alcohol such as sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, preferably in an amount of from +.0.1% w/v to 20% w/v, more preferably 1% w/v to 10% w/v, based on the total volume of the reagent.
As a preferred embodiment of the present application, the first buffer compound is preferably N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer.
As a preferred embodiment of the present application, the chaotropic compound is preferably one of a guanidine salt and a sodium salt, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, or a mixture of two or more thereof.
As a preferred technical scheme, the chelating agent-free complexing agent is preferably EDTA, EGTA, NTA (nitrilotriacetic acid), citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate and sodium alginate.
As a preferred embodiment of the present application, the chaotropic compound is preferably a guanidinium and sodium salt chaotropic compound, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, or a mixture of two or more thereof.
As a preferred embodiment of the present application, the second buffer compound is preferably N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer.
As a preferred embodiment of the present application, the nonionic surfactant is preferably a polyoxyethylene-based nonionic detergent, in particular a polyoxyethylene fatty acid ester, such as polyoxyethylene sorbitan monolaurate (tween-20), polyoxyethylene sorbitan monooleate (tween-80), polyoxyethylene alkylphenyl ethers, for example polyoxyethylene p-isooctylphenyl ether (triton X-100), polyoxyethylene tert-octylphenyl ether (triton X-114), polyoxyethylene isooctylphenyl ether (triton X-450), polyoxyethylene fatty alcohol ethers, such as polyoxyethylene (4) lauryl ether (Brij 30), polyoxyethylene (23) lauryl ether (Brij 35), polyoxyethylene (10) cetyl ether (Brij 56), polyoxyethylene (20) cetyl ether (Brij 58), polyoxyethylene stearyl ether, such as polyoxyethylene (2) stearyl ether (Brij 72).
As a preferred embodiment of the present application, the alcohol is preferably a branched and/or unbranched alcohol having 1 to 11 carbon atoms, most preferably selected from ethanol, ethylene glycol, isopropanol, n-butanol, 1, 3-butanediol.
As a preferred embodiment of the present application, the nucleic acid extraction specifically comprises the following steps:
s1: sample lysis: taking biological samples such as blood, tissues and semen, adding a lysis reagent and protease, mixing uniformly by vortex, incubating and lysing cells at 25-70 ℃ and releasing nucleic acid;
s2: nucleic acid adsorption: adding a binding solution into the cleavage mixture, then contacting the mixed solution with a solid material, adsorbing nucleic acid on the surface of the solid material, keeping other impurities in the solution, and discarding the liquid;
s3: cleaning a solid material: washing the solid material in a washing reagent, wherein nucleic acid is adsorbed on the surface of the solid material, and impurities such as protein, lipid and the like attached to the surface of the solid material are dissolved in the washing reagent and further removed;
s4: nucleic acid elution: the solid material is infiltrated by using a low-salt high-pH solution, and the nucleic acid can be released from the surface of the solid material into the eluent to complete the separation and purification of the nucleic acid.
The beneficial effects are that: compared with the prior art, the invention provides a cracking and washing reagent for extracting nucleic acid, which has the following beneficial effects: the cracking and washing reagent for extracting nucleic acid provides a novel cracking reagent, which further improves the capability of cracking biological samples to release nucleic acid; the novel washing reagent is provided, so that the washing capability on the surface of a solid material is further improved, the purity of the extracted nucleic acid is improved, the downstream application inhibition is smaller, and the nucleic acid detection sensitivity can be effectively improved;
the patent provides a lysis reagent for nucleic acid extraction,
no chaotropic compound is contained. The chaotropic compounds are a class of compounds that can disrupt the hydrogen bond network between water molecules, having a denaturing effect on proteins, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, and/or mixtures of two or more thereof.
No chelating or complexing agent is contained. Non-limiting examples of such agents are EDTA, EGTA, NTA (nitrilotriacetic acid), citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate, sodium alginate.
At least one surfactant is included, which is an anionic surfactant, preferably a sulphate or sulphonate salt of a fatty alcohol, in particular a bile acid based detergent, such as sodium deoxycholate, sodium cholate, a sarcosine based detergent, such as sarcosyl sarcosine, sodium N-lauroyl sarcosine, a sulphate or sulphonate salt of a fatty alcohol, such as sodium dodecyl sulphate, sodium dodecyl sulphate or sodium dodecyl benzene sulphonate. The amount is preferably from ∈ 0.1% w/v to 20% w/v, more preferably from 1% w/v to 10% w/v, based on the total volume of the reagent.
Comprising at least one buffering compound, preferably selected from the group consisting of: n- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS), and/or phosphate buffers.
The patent provides a washing reagent for nucleic acid extraction,
at least one chaotropic compound, preferably a guanidinium or sodium salt, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate and/or a mixture of two or more thereof.
Comprising at least one buffer compound, preferably selected from: n- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS), and/or phosphate buffers.
Comprises at least one nonionic surfactant, preferably a polyoxyethylene-based nonionic detergent, in particular a polyoxyethylene fatty acid ester, such as polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene sorbitan monooleate (Tween-80), polyoxyethylene alkylphenyl ethers, such as polyoxyethylene p-isooctylphenyl ether (triton X-100), polyoxyethylene tert-octylphenyl ether (triton X-114), polyoxyethylene isooctylphenyl ether (triton X-450), polyoxyethylene fatty alcohol ethers, such as polyoxyethylene (4) lauryl ether (Brij 30), polyoxyethylene (23) lauryl ether (Brij 35), polyoxyethylene (10) cetyl ether (Brij 56), polyoxyethylene (20) cetyl ether ]58 Polyoxyethylene stearyl ether such as polyoxyethylene (2) stearyl ether (Brij 72).
The nonionic surfactant is preferably used in an amount of from ∈0.01wt/vol to 10wt/vol, more preferably from 0.1wt/vol to 1wt/vol.
At least one alcohol, preferably from branched and/or unbranched alcohols having 1 to 11 carbon atoms, most preferably from ethanol, ethylene glycol, isopropanol, n-butanol, 1, 3-butanediol, the whole lysis and washing reagent being simple in structure, convenient to operate and better in use than in the conventional manner.
Drawings
FIG. 1 is a schematic diagram showing an embodiment of a lysis and washing reagent for nucleic acid extraction according to the present invention.
FIG. 2 is a schematic diagram of a second embodiment of a lysis and washing reagent for nucleic acid extraction according to the present invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the 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.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
As shown in fig. 1 and 2, a lysis and washing reagent for nucleic acid extraction, wherein the lysis solution comprises a surfactant, a first buffer compound, a chaotropic compound and a chelating complexing agent-free, and the washing reagent comprises a chaotropic compound, a second buffer compound, a nonionic surfactant and alcohol, so that a novel lysis reagent is provided, and the nucleic acid release capacity of a lysed biological sample is further improved; the novel washing reagent is provided, the washing capability of the surface of a solid material is further improved, the purity of the extracted nucleic acid is improved, the inhibition to downstream application is smaller, and the nucleic acid detection sensitivity can be effectively improved.
Further, the surfactant is preferably a sulfate or sulfonate of a fatty alcohol, in particular a bile acid based detergent such as sodium deoxycholate, sodium cholate, a sarcosine based detergent such as sarcosyl sarcosine, sodium N-lauroyl sarcosine, a sulfate or sulfonate of a fatty alcohol such as sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, preferably in an amount of from +.0.1% to 20% weight/volume, more preferably 1% to 10% weight/volume based on the total volume of the reagent.
Further, the first buffer compound is preferably N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer.
Further, the chaotropic compound is preferably one of a guanidine salt and a sodium salt, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, or a mixture of two or more thereof.
Further, the chelating agent-free agent is preferably EDTA, EGTA, NTA (nitrilotriacetic acid), citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate, or sodium alginate.
Further, the chaotropic compound is preferably a guanidinium and sodium salt chaotropic compound such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, or a mixture of two or more thereof.
Further, the second buffer compound is preferably N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer.
Further, the nonionic surfactant is preferably a polyoxyethylene-based nonionic detergent, in particular a polyoxyethylene fatty acid ester such as polyoxyethylene sorbitan monolaurate (tween-20), polyoxyethylene sorbitan monooleate (tween-80), polyoxyethylene alkylphenyl ethers such as polyoxyethylene p-isooctylphenyl ether (triton X-100), polyoxyethylene tert-octylphenyl ether (triton X-114), polyoxyethylene isooctylphenyl ether (triton X-450), polyoxyethylene fatty alcohol ethers such as polyoxyethylene (4) lauryl ether (Brij 30), polyoxyethylene (23) lauryl ether (Brij 35), polyoxyethylene (10) cetyl ether (Brij 56), polyoxyethylene (20) cetyl ether (Brij 58), polyoxyethylene stearyl ether such as polyoxyethylene (2) stearyl ether (Brij 72).
Further, the alcohol is preferably a branched and/or unbranched alcohol having 1 to 11 carbon atoms, most preferably selected from ethanol, ethylene glycol, isopropanol, n-butanol, 1, 3-butanediol.
Further, the nucleic acid extraction specifically comprises the following steps:
s1: sample lysis: taking biological samples such as blood, tissues and semen, adding a lysis reagent and protease, mixing uniformly by vortex, incubating and lysing cells at 25-70 ℃ and releasing nucleic acid;
s2: nucleic acid adsorption: adding a binding solution into the cleavage mixture, then contacting the mixed solution with a solid material, adsorbing nucleic acid on the surface of the solid material, keeping other impurities in the solution, and discarding the liquid;
s3: cleaning a solid material: washing the solid material in a washing reagent, wherein nucleic acid is adsorbed on the surface of the solid material, and impurities such as protein, lipid and the like attached to the surface of the solid material are dissolved in the washing reagent and further removed;
s4: nucleic acid elution: the solid material is infiltrated by using a low-salt high-pH solution, and the nucleic acid can be released from the surface of the solid material into the eluent to complete the separation and purification of the nucleic acid.
The lysate comprises:
at least one surfactant, the molecular planar structure of which is known to be anionic, has one polar and one non-polar face, has a high CMC, small micelles, and has excellent protein denaturation and dissolution capacity, preferably is a sulfate or sulfonate of a fatty alcohol, in particular a bile acid-based detergent, such as sodium deoxycholate, sodium cholate, a sarcosine-based detergent, such as sarcosyl, N-lauroyl sarcosyl, a sulfate or sulfonate of a fatty alcohol, such as sodium dodecyl sulfate, sodium dodecyl sulfonate or sodium dodecyl benzene sulfonate. The amount is preferably from ∈ 0.1% w/v to 20% w/v, more preferably from 1% w/v to 10% w/v, based on the total volume of the reagent.
At least one buffering compound, preferably selected from: n- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS), and/or phosphate buffers.
No chaotropic compound is contained. Chaotropic compounds are compounds which can break the hydrogen bond network between water molecules and have denaturation effect on proteins. Chaotropic compounds commonly used in the art are guanidine salts or sodium salts, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate and/or mixtures of two or more thereof.
No chelating or complexing agent is contained. Non-limiting examples of such agents are EDTA, EGTA, NTA (nitrilotriacetic acid), citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate, sodium alginate.
The washing reagent comprises:
at least one chaotropic compound, according to the Hofmeister series (Hofmeister series), suitable chaotropic compounds, preferably guanidinium or sodium salt chaotropic compounds, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate and/or mixtures of two or more thereof, can be found.
At least one buffer compound, preferably selected from: n- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS), and/or phosphate buffers.
At least one nonionic surfactant, preferably a polyoxyethylene-based nonionic detergent, in particular a polyoxyethylene fatty acid ester, such as polyoxyethylene sorbitan monolaurate (tween-20), polyoxyethylene sorbitan monooleate (tween-80), polyoxyethylene alkylphenyl ethers, such as polyoxyethylene p-isooctylphenyl ether (triton X-100), polyoxyethylene tert-octylphenyl ether (triton X-114), polyoxyethylene isooctylphenyl ether (triton X-450), polyoxyethylene fatty alcohol ethers, such as polyoxyethylene (4) lauryl ether (Brij 30), polyoxyethylene (23) lauryl ether (Brij 35), polyoxyethylene (10) cetyl ether (Brij 56), polyoxyethylene (20) cetyl ether @58 Polyoxyethylene stearyl ether such as polyoxyethylene (2) stearyl ether (Brij 72).
At least one alcohol, preferably from branched and/or unbranched alcohols having 1 to 11 carbon atoms, most preferably selected from ethanol, ethylene glycol, isopropanol, n-butanol, 1, 3-butanediol.
Example 1
Preparing a cracking reagent 1, wherein the formula comprises the following components:
the following weight percentages of reagents were weighed: 40% -70% of guanidine isothiocyanate, 5% -15% of deoxycholate sodium, 0.1% -1% of EDTA and 0.1% -1% of Tris-base, adding a proper amount of purified water for dissolution, and adjusting the pH to 8.0 by using hydrochloric acid.
Preparing a washing reagent 1, wherein the formula comprises the following components:
the following weight percentages of reagents were weighed: 50% -70% guanidine hydrochloride, 0.1% -1% sodium citrate and 30% -50% ethanol, adding a proper amount of purified water for dissolution, and adjusting the pH to 6.0 by using citric acid solution.
Nucleic acids were extracted from 16 tissue samples using lysis reagent 1, binding reagent, washing reagent 1, magnetic beads, elution reagent. The extraction operation follows the following steps:
1) Sample lysis: placing 20mg of tissue sample into a 1.5mL centrifuge tube, adding a lysis reagent and proteinase K, mixing uniformly by vortex, and incubating at 60 ℃ until the tissue sample is completely digested and dissolved;
2) Nucleic acid adsorption: binding reagents, magnetic beads, are added to the lysis mixture and vortexed to bind the nucleic acids to the surface of the magnetic beads.
3) First magnetic bead cleaning: separating magnetic beads, discarding liquid, adding washing reagent, vortex mixing, and washing magnetic beads.
4) Drying magnetic beads:
5) Nucleic acid elution: separating magnetic beads, discarding liquid, adding nucleic acid eluting reagent, and vortex mixing to release nucleic acid from the surface of magnetic beads into the eluent.
The amounts of DNA extracted from samples No. 1-16 were measured optically by Nanodrop Siemens technology (Thermo Scientific), and the results are shown in Table 1.
As a result, it was found that using a lysis reagent containing an chaotropic compound, a chelating agent and a washing reagent containing no surfactant, a part of the sample was extracted with a nucleic acid 260/280 of less than 1.8, a nucleic acid 260/230 of less than 2.0, and a nucleic acid 260/280 of less than that specified in the current standard GB/T37874-2019 should be 1.7-19, and a nucleic acid 260/230 should be greater than 2.0. The purity of the extracted nucleic acid is low.
DNA electrophoresis was performed on samples 1 to 16 on a 1% agarose gel, and the results are shown in FIG. 1.
As a result, it was found that part of the nucleic acid accumulated in the agarose gel sample-loading well and could not enter the gel under the action of the electric field, indicating insufficient lysis of the tissue sample and crosslinking of part of the nucleic acid with proteins or cell debris.
Example 2
Preparing a cracking reagent 2, wherein the formula comprises the following components:
the following weight percentages of reagents were weighed: 5% -15% of deoxycholate sodium and 0.1% -1% of Tris-base, adding a proper amount of purified water for dissolution, and adjusting the pH value to 8.0 by using hydrochloric acid.
Preparing a washing reagent 2, wherein the formula comprises the following components:
the following weight percentages of reagents were weighed: 50% -70% of guanidine hydrochloride, 0.1% -1% of sodium citrate, 30% -50% of ethanol and 0.1% -1% of NP-40, adding a proper amount of purified water for dissolution, and adjusting the pH to 6.0 by using a citric acid solution.
Nucleic acids were extracted from 16 tissue samples using lysis reagent 1, binding reagent, washing reagent 1, magnetic beads, elution reagent. The extraction operation follows the following steps:
1) Sample lysis: placing 20mg of tissue sample into a 1.5mL centrifuge tube, adding a lysis reagent and proteinase K, mixing uniformly by vortex, and incubating at 60 ℃ until the tissue sample is completely digested and dissolved;
2) Nucleic acid adsorption: binding reagents, magnetic beads, are added to the lysis mixture and vortexed to bind the nucleic acids to the surface of the magnetic beads.
3) First magnetic bead cleaning: separating magnetic beads, discarding liquid, adding washing reagent, vortex mixing, and washing magnetic beads.
4) Drying magnetic beads:
5) Nucleic acid elution: separating magnetic beads, discarding liquid, adding nucleic acid eluting reagent, and vortex mixing to release nucleic acid from the surface of magnetic beads into the eluent.
The amounts of DNA were measured optically by Nanodrop Siemens technology company (Thermo Scientific), and the results are shown in Table 2.
As a result, it was found that the use of a cleavage reagent free of a chaotropic compound, a chelating agent and a detergent reagent containing a surfactant resulted in the extraction of nucleic acids 260/280 between 1.8 and 1.9, 260/230 greater than 2.0, and 260/280 in compliance with the current standard GB/T37874-2019 should be between 1.7 and 19, and 260/230 should be greater than 2.0. The purity of the extracted nucleic acid meets the requirement.
The amounts of DNA were measured optically by 1% agarose gel electrophoresis, respectively, and the results are shown in FIG. 2.
As a result, the nucleic acid bands in the agarose gel are clear, no nucleic acid is accumulated at the sample-adding hole, which indicates that the tissue sample is sufficiently cracked, and no obvious protein or cell debris exists in the nucleic acid eluent.
It should be noted that in this document, relational terms such as first and second (first and second), and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims.

Claims (10)

1. A lysis and wash reagent for nucleic acid extraction, characterized in that: the lysate comprises a surfactant, a first buffer compound, a chaotropic compound and a chelating complexing agent, and the washing reagent comprises a chaotropic compound, a second buffer compound, a nonionic surfactant and an alcohol.
2. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the surfactant is preferably a sulfate or sulfonate of a fatty alcohol, in particular a bile acid-based detergent such as sodium deoxycholate, sodium cholate, a sarcosine-based detergent such as sarcosyl sarcosine, sodium N-lauroyl sarcosine, a sulfate or sulfonate of a fatty alcohol such as sodium dodecyl sulfate, sodium dodecyl sulfonate, sodium dodecyl benzene sulfonate, preferably in an amount of from +.0.1% to 20% weight/volume, more preferably from 1% weight/volume to 10% weight/volume, based on the total volume of the reagent.
3. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the first buffer compound is preferably N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer.
4. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the chaotropic compound is preferably one of a guanidine salt and a sodium salt, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, or a mixture of two or more thereof.
5. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the chelating agent-free complexing agent is preferably EDTA, EGTA, NTA (nitrilotriacetic acid), citric acid, iminodisuccinic acid, isoascorbic acid, triethanolamine, tartaric acid, sodium gluconate, or sodium alginate.
6. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the chaotropic compound preferably has a guanidinium salt and a sodium salt chaotropic compound, such as sodium acetate, sodium iodide, sodium perchlorate, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, or a mixture of two or more thereof.
7. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the second buffer compound is preferably N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), TRIS (hydroxymethyl) aminomethane (TRIS), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-bis (2-hydroxyethyl) glycine (BICINE), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer.
8. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the nonionic surfactant is preferably a polyoxyethylene-based nonionic detergent, in particular a polyoxyethylene fatty acid ester, such as polyoxyethylene sorbitan monolaurate (tween-20), polyoxyethylene sorbitan monooleate (tween-80), polyoxyethylene alkylphenyl ethers, for example polyoxyethylene p-isooctylphenyl ether (triton X-100), polyoxyethylene tert-octylphenyl ether (triton X-114), polyoxyethylene isooctylphenyl ether (triton X-450), polyoxyethylene fatty alcohol ethers, such as polyoxyethylene (4) lauryl ether (Brij 30), polyoxyethylene (23) lauryl ether (Brij 35), polyoxyethylene (10) cetyl ether (Brij 56), polyoxyethylene (20) cetyl ether (Brij 58), polyoxyethylene stearyl ether, such as polyoxyethylene (2) stearyl ether (Brij 72).
9. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the alcohol is preferably a branched and/or unbranched alcohol having 1-11 carbon atoms, most preferably selected from ethanol, ethylene glycol, isopropanol, n-butanol, 1, 3-butanediol.
10. The cleavage and washing reagent for nucleic acid extraction according to claim 1, wherein: the nucleic acid extraction specifically comprises the following operation steps:
s1: sample lysis: taking biological samples such as blood, tissues and semen, adding a lysis reagent and protease, mixing uniformly by vortex, incubating and lysing cells at 25-70 ℃ and releasing nucleic acid;
s2: nucleic acid adsorption: adding a binding solution into the cleavage mixture, then contacting the mixed solution with a solid material, adsorbing nucleic acid on the surface of the solid material, keeping other impurities in the solution, and discarding the liquid;
s3: cleaning a solid material: washing the solid material in a washing reagent, wherein nucleic acid is adsorbed on the surface of the solid material, and impurities such as protein, lipid and the like attached to the surface of the solid material are dissolved in the washing reagent and further removed;
s4: nucleic acid elution: the solid material is infiltrated by using a low-salt high-pH solution, and the nucleic acid can be released from the surface of the solid material into the eluent to complete the separation and purification of the nucleic acid.
CN202311503582.0A 2023-11-13 2023-11-13 Cleavage and washing reagent for nucleic acid extraction Pending CN117402872A (en)

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