CN113265399A - Green and rapid animal tissue DNA extraction method - Google Patents
Green and rapid animal tissue DNA extraction method Download PDFInfo
- Publication number
- CN113265399A CN113265399A CN202110707478.8A CN202110707478A CN113265399A CN 113265399 A CN113265399 A CN 113265399A CN 202110707478 A CN202110707478 A CN 202110707478A CN 113265399 A CN113265399 A CN 113265399A
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- Prior art keywords
- dna
- animal tissue
- tris
- solution
- acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- 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
- C12N15/1013—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 by using magnetic beads
Abstract
The invention relates to a method for separating and/or purifying DNA of animal tissue and an extraction kit. The method has the advantages that: 1. the operation is simple and time-saving, and is finished in about 15 minutes; 2. the environment is protected, the health of experimenters is facilitated, and the environment is not polluted; 3. the cost is low, and all the used reagents are cheap; 4. the quality and quantity of the extracted DNA are good.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to a method for separating and/or purifying DNA of animal tissue and an extraction kit.
[ background of the invention ]
With the development of genome sequencing technology, the sequence, structure and function research of biological genome is rapidly developed, and obtaining biological genome DNA with high purity, high content and high integrity is the first prerequisite for the application of genome sequencing technology. The traditional biological genome DNA extraction method mainly comprises a CTAB method, a phenol method and a CTAB method, and the extraction steps mainly comprise the steps of carrying out primary treatment on biological tissues and then carrying out processes of cell lysis, impurity removal, DNA precipitation, rinsing, elution and the like. It mainly uses CTAB or CTAB detergent to crack the cell, to release the genome DNA in the cell; then removing impurities such as protein, RNA and the like by a method of chloroform and phenol extraction or high-salt precipitation; then adding a proper volume of organic solvent such as absolute ethyl alcohol, isopropanol or PEG and the like into the extracted supernatant to precipitate or adsorb the DNA on media such as a silica gel column, an ion exchange column and the like; finally, after rinsing with a rinsing solution, the solution is dissolved by a low-concentration salt solution or eluted from the medium.
The existing DNA extraction method is often accompanied with protein, salt, impurity and other pollution, and has the disadvantages of complicated steps and more time-consuming extraction process. Such as the sodium perchlorate method, the urea method, and the like. Meanwhile, the methods require a large sample amount, and organic matters such as phenol and the like are easy to remain in DNA solution, and particularly interfere with the amplification effect in the subsequent PCR amplification process, so that the PCR result is influenced, and the application of the methods is greatly limited.
The current common animal tissue DNA extraction method comprises the following steps: (a) chloroform, isoamyl alcohol and phenol extraction method are adopted to remove protein, salt and other impurities. Phenol is a denaturant in DNA extraction processes, and the use of chloroform in combination with repeated extraction can yield DNA of relatively high quality. However, phenol and chloroform are toxic to human bodies and are not beneficial to the physical health and environmental protection of experimenters. The whole extraction process is complicated in steps and long in time consumption, and DNA degradation is often caused easily. (b) Both high temperature incubation and low temperature placement increase the cost of DNA extraction. (c) DNA extraction is very time-consuming, usually around 1 hour.
[ summary of the invention ]
It is an object of the present invention to provide a method and an extraction kit that overcome at least one of the above-mentioned drawbacks of the prior art. The kit comprises but is not limited to an animal tissue and/or cell green rapid DNA extraction kit, a blood green rapid DNA extraction kit, a saliva green rapid DNA extraction kit, an oral cell green rapid DNA extraction kit and the like.
In order to achieve the above object, the present invention provides in a first aspect a method for extracting DNA from animal tissue, comprising the steps of:
(a) treating animal tissue;
(b) transferring the treated animal tissue into the first solution, shaking up, adding the second solution, and standing for 1-10 minutes, preferably for 2-5 minutes, more preferably for 2 minutes;
(c) fixing animal tissue DNA on a substrate, adding branched or unbranched alkanol with the volume of 0.4-0.9 times of the volume of supernatant, uniformly mixing, and transferring to a centrifugal column or a magnetic ball, and the like;
(d) washing and eluting the DNA;
in a preferred embodiment, the composition of the first solution is:
-mixtures containing at least cetylammonium bromide (CTAB) and/or with other surfactants, wherein the CTAB concentration is between 1% and 6%;
-at least one or more chelating agent compounds, preferably selected from, including but not limited to: N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N, N '-disuccinic acid (EDDS), 1, 2-bis (o-aminophenoxy) ethane-N, N' -tetraacetic acid (BAPTA), and phosphonate chelating agents (including, for example and without limitation, nitrilotris (methylene) phosphonic acid (NTMP), ethylenediaminetetra (methylene phosphonic acid) (EDTMP), diethylenetriaminepenta (methylene) phosphonic acid (DTPMP), 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP), etc.), preferably EDTA, at a concentration of 20mM to 300 mM;
-at least one salt solution or a mixture of more than one salt solution, preferably NaCl, in a concentration of 1M to 3M;
-and/or one or more buffer compounds, the pH value of which may be between 3 and 11, preferably selected from, but not limited to: TRIS (hydroxymethyl) aminomethane (TRIS), N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), N-bis (2-hydroxyethyl) glycine (BICINE), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer, sodium acetate buffer, methyl acetate buffer, etc., preferably ph8.0tris-HCl, at a concentration of 20mM to 300mM, more preferably at a concentration of 100mM to 200 mM;
-and/or one or more water-soluble organic substances, preferably selected from, including but not limited to: guanidine hydrochloride, guanidine isothiocyanate, glycerol, glucose, fructose, sucrose, maltose and the like, preferably glycerol, in a concentration of: 1% -10%;
and/or one or more other substances which have little or no effect.
In a preferred embodiment, the second solution consists of:
-2-5M of a salt solution or salt mixture solution having a pH of 3-11, preferably 5M sodium chloride.
For the purposes of the present invention, the term "salt" is intended to mean a class of metal ions or ammonium ions (NH)4 +) Compounds formed by ionic bonding with acid ions (anions) dissociate all ions when dissolved in water. Specifically, inorganic salt compounds, such as, but not limited to: sodium chloride, potassium nitrate, potassium acetate, potassium sulfate, potassium iodide, sodium nitrate, sodium acetate, sodium sulfate, sodium bromide, sodium iodide, sodium perchlorate, ammonium chloride, ammonium nitrate, ammonium sulfate, and/or mixtures of two or more thereof, and the like.
For the purposes of the present invention, the term "buffer compound" denotes compounds which can provide a buffering or pH-stabilizing effect to an aqueous solution.
For the purposes of the present invention, the term "DNA" is intended to mean genetic material, such as nuclear DNA, mitochondrial DNA, and the like.
For the purposes of the present invention, the term "surfactant" denotes a substance capable of causing a significant reduction in the surface tension of a target solution, and can be divided into: the ionic surfactant comprises cationic surfactant, anionic surfactant, nonionic surfactant, amphoteric surfactant, compound surfactant, other surfactants and the like.
For the purposes of the present invention, the term "chelating agent" denotes compounds capable of chelating metal ions, such as EDTA and the like.
For the purposes of the present invention, the information "% weight/volume", "% (weight/volume)" or "% (w/v)" represents information, for example, in grams of surfactant per 100 milliliters of reagent or composition.
According to a preferred embodiment of the invention, the salt solution is an inorganic salt solution, preferably selected from: sodium chloride, sodium acetate, ammonium chloride, ammonium nitrate, ammonium sulfate, sodium iodide, sodium perchlorate and/or mixtures of more than one salt.
According to a preferred embodiment of said method, and/or one or more buffer compounds selected from the group consisting of: TRIS (hydroxymethyl) aminomethane (TRIS), N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), N-bis (2-hydroxyethyl) glycine (BICINE), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffers, sodium acetate, methyl acetate and the like
According to a particularly preferred embodiment of the method, and/or a buffer compound selected from: TRIS (hydroxymethyl) aminomethane (TRIS) and/or N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES) and/or sodium acetate and/or phosphate buffer. According to yet a more preferred embodiment of the method, the lysis and/or binding composition comprises at least one buffer compound selected from the group consisting of: TRIS (hydroxymethyl) aminomethane (TRIS) and/or sodium acetate. Preferably, PH8.0100mM to 300mM Tris-HCl is selected.
According to a further preferred embodiment, the pH of the solution is between 3 and 11, preferably between 4.5 and 8.
In a preferred embodiment, in particular the solution may further comprise enzymes such as lyase, RNase a, in particular, such as proteinase K, protease, zymolase, achromopeptidase, cytolytic enzyme, lysostaphin, lysozyme, ribozymes and the like.
The DNA is immobilized onto a substrate in the presence of a salt solution, preferably a branched or unbranched alkanol, including but not limited to: centrifugal column and magnetic ball. Accordingly, binding agents comprising at least one branched or unbranched alkanol are preferred.
Preferably useful are short chain branched or unbranched alkanols having 1 to 5 carbon atoms. According to a preferred embodiment of the invention, the branched or unbranched alkanol is an alcohol having 1 to 5 carbon atoms, preferably selected from: methanol, ethanol, n-propanol, isopropanol, branched or unbranched butanol or pentanol, and/or mixtures thereof.
Unless otherwise indicated, the definition of "branched or unbranched alkanol", in particular propanol, butanol and pentanol, includes the isomeric forms of any of the particular groups which may be consumed. Thus, for example, branched or unbranched propanols include n-propanol and isopropanol, branched or unbranched butanols include n-butanol, isobutanol, sec-butanol, and tert-butanol, and branched or unbranched pentanols include, for example, n-pentanol and isopentanol. Preference is given to using alcohols selected from the group consisting of: methanol, ethanol, isopropanol, and/or mixtures thereof, it is particularly preferred to use an alcohol selected from the group consisting of: ethanol, isopropanol, and/or mixtures thereof.
According to a preferred embodiment, the branched and/or unbranched alcohol comprises from 0.1 to 1 times the volume of the supernatant, preferably from 0.3 to 0.8 times, and more preferably from 0.5 to 0.7 times the volume of the supernatant.
The DNA extraction of the animal tissue sample may be performed at room temperature, e.g.between 15 ℃ and 25 ℃, or at elevated temperature, e.g.between 30 ℃ and 90 ℃.
According to a preferred embodiment of the invention, an incubation time of from 10 seconds to 30 minutes is convenient for DNA. According to the method described above, a particularly preferred embodiment, an incubation time of from 1 minute to 5 minutes is convenient for DNA. According to the above method, according to yet a more preferred embodiment, an incubation time of about 3 minutes is advantageous.
According to a preferred embodiment of the invention, the DNA is isolated by contacting the sample with a matrix based on one or more silica compounds, such as silica, silicates, glass and/or silica gel, and incubating for a time sufficient to effect binding. The matrix may be of conventional design known in the art, e.g. in the form of particles, membranes or filters, etc. For ease of removal, particles with magnetic properties are preferred.
It is preferred to have magnetic particles with a colloidal silica coating to isolate the DNA. The DNA is preferably isolated using magnetic particles having a colloidal silica coating and an average particle size in the range of 1um to 40um, preferably between 5um and 20um, particularly preferably between 6um and 10um, preferably with a narrow particle size distribution. More preferably, magnetic particles with a colloidal silica coating and an average particle size between 6um and 10um, preferably a narrow size distribution, are used to separate DNA.
In a further preferred embodiment, the magnetic or magnetically attractable particles are particles with iron oxide based magnetism, preferably selected from magnetite (Fe3O4), maghemite (γ -Fe2-O3) and/or ferrite.
Magnetic silica particles which can be used in an advantageous manner can be found, for example, in international application WO 01/71732, the contents of which are incorporated herein by reference.
In a preferred embodiment, a matrix based on one or more silica compounds in the form of magnetically or magnetically attractable particles with a silica surface can be used.
The bonding is carried out at a temperature of between 4 ℃ and 90 ℃, preferably between 20 ℃ and 70 ℃, particularly preferably between 45 ℃ and 70 ℃, most particularly preferably between 50 ℃ and 65 ℃. The bonding can also be carried out at room temperature, for example between 15 ℃ and 25 ℃.
After incubation, the nucleic acids bound to the matrix mixture based on one matrix or more than one silica compound are removed from the lysis and/or binding composition. When using magnetic silica particles, this can be achieved with the aid of a magnetic field. For example, by applying a magnetic field to drag magnetic particles onto the walls of the vessel in which incubation is taking place, the liquid is removed with a pipette tip or a pipette gun; or fixing the magnetic particles on a magnetic rod protected by a plastic coating, and taking out the magnetic rod to remove the waste liquid.
Preferably, the DNA immobilized on the substrate may be washed prior to removal. The washing step is preferably performed by incubating the wash solution with the loaded particles, preferably involving resuspension of the particles, e.g. by shaking or application of a magnetic field. Preferably a decontamination of the washing solution.
The wash reagent used may be a conventional wash buffer or any other suitable medium. Generally, washing reagents having low to moderate ionic strength are preferred, such as a solution of 10mM TRIS (hydroxymethyl) aminomethane (TRIS) and/or EDTA and the like and/or a 0.05M to 0.2M sodium citrate solution and the like. Wash buffers with higher salt concentrations, such as 4-6M guanidine hydrochloride solutions, may also be used. As noted above, the wash reagents of the invention are similarly suitable wash reagents.
Also, an alcohol-containing washing agent, for example, an aqueous solution of an alcohol having 1 to 5 carbon atoms, preferably an aqueous solution of ethanol, specifically an aqueous solution containing 50 to 100% ethanol, may be used.
The DNA immobilized to the substrate is preferably washed several times, e.g.1 to 4 times, preferably with different washing reagents. In a preferred embodiment, the washing is first carried out with a washing reagent having a low to moderate ionic strength and then the DNA is washed again with an aqueous solution containing 70-100% ethanol.
More specifically, with magnetic particles, the separation and/or washing steps are facilitated due to the magnetic aggregation of the particles.
After the final washing step or water washing, the preferred magnetic particles may be dried, for example, vacuum dried or by evaporating the liquid or allowing the liquid to evaporate.
The bound DNA may be removed from the matrix. The process of removing DNA is called elution.
Bound DNA can be removed from the particles by means of an elution reagent with a low salt content. More specifically, a reagent having a salt content of less than 0.1M may be used as the elution reagent having a low salt content. Particular preference is given to elution reagents comprising the buffer compound TRIS (hydroxymethyl) aminomethane (TRIS). Also particularly suitable for elution is demineralized water, optionally containing one or more additives, for example chelating agents such as ethylenediaminetetraacetic acid (EDTA), azide compounds and/or buffer compounds such as TRIS (hydroxymethyl) aminomethane (TRIS).
In a preferred embodiment, the kit further comprises a matrix based on one or more silica compounds. In particular a matrix based on one or more silica compounds in the form of magnetically or magnetically attractable particles having a silica surface. Examples of magnetic silica particles preferably comprised in the kit are described in international application WO 01/71732, the entire content of which is incorporated herein by reference.
In a further preferred embodiment, the kit may comprise a silanized carrier material in addition to the magnetic silica particles, preferably a spin column with a silica membrane.
The detergent used may be a conventional buffer or any other suitable medium. Generally, detergents having low to moderate ionic strength are preferred, such as a solution of 10mM TRIS (hydroxymethyl) aminomethane (TRIS) and/or EDTA and the like and/or a 0.05M to 0.2M sodium citrate solution and the like. A wash buffer having a higher salt concentration, such as a 4-6M solution of guanidine hydrochloride, may also be used. As noted above, the wash reagents of the invention are similarly suitable wash reagents.
Also, an alcohol-containing washing agent, for example, an aqueous solution of an alcohol having 1 to 5 carbon atoms, preferably an aqueous solution of ethanol, specifically an aqueous solution containing 50 to 100% ethanol, may be used.
The DNA immobilized to the spin column is preferably washed several times, e.g.2-3 times, preferably with different washing reagents. In a preferred embodiment, the washing is first carried out with a washing reagent having a low to moderate ionic strength, and then the DNA is washed again with an aqueous solution containing 70-100% ethanol.
According to the embodiments, bound DNA may be removed from the spin column. The process of removing DNA is called eluting DNA. Bound DNA can be removed from the spin column by means of an elution reagent with a low salt content. More specifically, a reagent having a salt content of less than 0.1M may be used as the elution reagent having a low salt content. Particular preference is given to elution reagents comprising the buffer compound TRIS (hydroxymethyl) aminomethane (TRIS). Also particularly suitable for elution is demineralized water, optionally containing one or more additives, for example chelating agents such as ethylenediaminetetraacetic acid (EDTA), azide compounds and/or buffer compounds such as TRIS (hydroxymethyl) aminomethane (TRIS).
According to a preferred embodiment, the elution reagent is eluted once or more, preferably 1 to 3 times, more preferably 1 time.
The carrier for describing the method for rapidly extracting the animal tissue DNA is typically a paper specification, and may be any carrier (including but not limited to a removable disk, an optical disk, an electronic ink screen, a network resource, an address thereof, and the like) for describing an electronic version of the method, as long as the method can be known by reading the carrier, and the carrier is within the scope of the concept.
The present invention provides in a further aspect a computer readable carrier carrying a computer program comprising instructions for carrying out the aforementioned lysis, binding, washing and/or elution reagents. The computer is understood in a broad sense and includes but is not limited to a single chip microcomputer, a PLC, a single chip microcomputer, an industrial personal computer, a PC and the like. The computer readable carrier includes, but is not limited to, any form of Flash, EEPROM, magnetic disk (floppy or hard disk), optical disk, and the like. The computer program may be written in any language, such as assembly, JAVA, VB, VC, C + +, Python, as long as the associated system is controlled to implement the method.
The method and the kit for rapidly extracting the DNA of the animal tissue, which are provided by the invention, can at least produce the following beneficial effects:
1. the operation is simple, the time is saved, and if the DNA extraction is carried out within about 15 minutes, the sample treatment process is not included;
2. the cost is low, the DNA extraction can be completed at room temperature, the cost for maintaining high-temperature and low-temperature environments is not required, and the cost of the reagent provided by the method is low;
3. the quality and quantity of extracted DNA are very high;
4. the used reagents are nontoxic, which is beneficial to the health of experimenters and reduces the damage to the environment;
[ description of the drawings ]
FIG. 1 is a gel electrophoresis chart of the extraction results of NaCl with different concentrations in the method of the present invention;
FIG. 2 is a gel electrophoresis chart of CTAB extraction results of different concentrations in the method of the invention;
FIG. 3 is a gel electrophoresis chart of different isopropanol volume extraction results in the method of the present invention;
FIG. 4 is a gel electrophoresis chart of the blood volume extraction results of different snakeheads in the method of the invention;
FIG. 5 is a gel electrophoresis image of mouse liver DNA extracted in the method of the present invention;
[ detailed description ] embodiments
The invention is further described below in conjunction with the drawings and the specific embodiments, which are provided only to assist in understanding the invention.
Example 1 determination of the optimal NaCl concentration in the first solution
As previously described, take about 2X 106Cells were centrifuged at 12000rpm for one minute, the supernatant was discarded, 320ul of a protective solution (10mM Tris-HCl, 1mM EDTA) was added, the cells were subjected to freeze-drying and grinding with liquid nitrogen, and then, 500ul of a first solution (3% CTAB, 3% glycerol, 20mM EDTA, PH8.0100mM Tris-HCl, 8ul of 10mg/ml RNase A was added temporarily) and NaCl at concentrations of 1.6M, 1.8M, 2.0M, 2.2M, 2.4, and 2.6M, respectively, were shaken well, 75ul of a second solution (5M NaCl) was added thereto, shaken well, left for 2 minutes, 12000rpm, centrifuged for one minute, the supernatant was taken out into a new EP tube, isopropanol was added in an amount of 0.6 times the volume of the supernatant was added, shaken well, centrifuged, washed for 2 times, left to stand at room temperature for 2 minutes, and DNA was eluted from the column with 100ul of an eluent. The whole DNA extraction process is completed at room temperature, and the time is within about 10 minutes.
The results were measured on a biaosharp (table 1) and examined by gel electrophoresis (fig. 1), which showed better results with 1.6-2.6M NaCl, the best 2.0M NaCl, and more intact and less degraded fragments of cellular genomic DNA.
Table 1: cell DNA quality extracted at different NaCl concentrations
| Concentration of NaCl | 1.6M | 1.8M | 2.0M | 2.2M | 2.4M | 2.6M |
| DNA concentration | 64.18ng/ul | 61.43ng/ul | 82.41ng/ul | 56.61ng/ul | 42.19ng/ul | 41.24ng/ul |
| A260/A280 | 1.847 | 1.835 | 1.829 | 1.843 | 1.833 | 1.830 |
Example 2 determination of optimal CTAB concentration in the first solution
As described above, about 2.4ml of human saliva was centrifuged at 12000rpm for one minute, the supernatant was discarded, 480. mu.l of a protective solution (10mM Tris-HCl, 1mM EDTA) was added, the cells were subjected to a liquid nitrogen freeze-drying and milling method, and then transferred to 300. mu.l of a first solution (2.0M NaCl, 3% glycerol, 20mM EDTA, PH8.0100mM Tris-HCl, and temporarily 8. mu.l of 10mg/ml RNase A) and CTAB at concentrations of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%, respectively, and shaken well, and then 45. mu.l of a second solution (5M NaCl) was added, shaken well, left for 2 minutes, 12000rpm for one minute, the supernatant was taken out into a new EP tube, isopropanol in an amount 0.6 times the volume of the supernatant was added, shaken well, transferred to a centrifugal column, washed 2 times, and left to stand at room temperature for 2 minutes, and the DNA was eluted from the column with 60. mu.l of an eluent. The whole DNA extraction process is completed at room temperature, and the time is within about 10 minutes.
The results were measured on a biaosharp (table 2) and examined by gel electrophoresis (figure 2), which showed better results for 0.5% to 3.0% CTAB, the best results for 2.5% CTAB, and fewer intact, degraded small fragments of salivary cell genomic DNA.
Table 2: quality of cellular DNA extracted at different CTAB concentrations
| CTAB concentration | 3％ | 2.5％ | 2.0％ | 1.5％ | 1.0％ | 0.5％ |
| DNA concentration | 78.23ng/ul | 89.72ng/ul | 63.18ng/ul | 52.61ng/ul | 66.84ng/ul | 48.65ng/ul |
| A260/A280 | 1.821 | 1.827 | 1.839 | 1.834 | 1.826 | 1.835 |
Example 3 determination of the optimum volume of Isopropanol
As described above, cells were taken from the oral cavity with a swab stick, placed in 1ml of a protective solution, shaken vigorously for about 30 seconds at 12000rpm, centrifuged for one minute, the supernatant was discarded, a total of 4 times, 320ul of the protective solution (10mM Tris-HCl, 1mM EDTA) was added, the oral cells were treated with liquid nitrogen freeze-drying grinding, then 300ul of the first solution (2.5% CTAB, 2.0M NaCl, 3% glycerol, 20mM EDTA, PH8.0100mM Tris-HCl, 8ul of 10mg/ml RNase A was added temporarily), shaken well, then 45ul of the second solution (5M NaCl) was added, shaken well, left for 2 minutes at 12000rpm, centrifuged for one minute, the supernatant was taken out and placed in a new EP tube, isopropanol was added in an amount of 0.5 to 0.8 times the volume of the supernatant, shaken well, centrifuged and transferred to a column, washed 2 times, left for 2 minutes at room temperature, and the DNA was eluted from the column with 60ul of the eluent. The whole DNA extraction process is completed at room temperature, and takes about 15 minutes.
The results were measured on a biaosharp (table 3) and examined by gel electrophoresis (fig. 3), which showed that 0.5V to 0.8V of isopropanol was preferable, and 0.6V of isopropanol was most preferable.
Table 3: quality of oral cell DNA extracted from different isopropanol volume ratios
| Volume of isopropyl alcohol | 0.5V | 0.6V | 0.7V | 0.8V |
| DNA concentration | 73.19ng/ul | 91.85ng/ul | 75.61ng/ul | 35.67ng/ul |
| A260/A280 | 1.843 | 1.848 | 1.841 | 1.838 |
Example 4 DNA extraction of snakehead blood of different volumes
As described above, a total of 280ul of fresh snakehead whole blood was taken, the blood was treated by liquid nitrogen freeze-drying milling to give 40ul, 60ul, 80ul and 100ul of 4 parts in proportion, then transferred to 300ul of the first solution (2.5% CTAB, 2.0M NaCl, 3% glycerol, 20mM EDTA, PH8.0100mM Tris-HCl, with 8ul of 10mg/ml RNase A temporarily added), shaken well, then added with 45ul of the second solution (5M NaCl), shaken well, left for 2 minutes, 12000rpm, centrifuged for one minute, the supernatant was taken out and put into a new EP tube, and isopropyl alcohol of 0.6 times the volume of the supernatant was added, shaken well, transferred to a centrifugal column, washed 2 times, left for 2 minutes at room temperature, and the DNA was eluted from the centrifugal column with 60ul of eluent. The whole DNA extraction process is completed at room temperature, and takes about 15 minutes.
The results were determined on a biaosharp (table 4) and examined by gel electrophoresis (fig. 4), which revealed that 80ul of snakehead blood was best for DNA extraction.
Table 4: extracting snakehead blood with different volumes to obtain DNA quality
| Volume of isopropyl alcohol | 0.5V | 0.6V | 0.7V | 0.8V |
| DNA concentration | 56.25ng/ul | 64.37ng/ul | 128.3ng/ul | 69.58ng/ul |
| A260/A280 | 1.817 | 1.823 | 1.834 | 1.831 |
Example 5 extraction of mouse liver tissue DNA
As described above, about 32mg of mouse heart was taken, mouse heart tissue was treated by liquid nitrogen freeze-drying milling, then 500ul of the first solution (2.5% CTAB, 2M NaCl, 3% glycerol, 20mM EDTA, PH8.0100mM Tris-HCl, 8ul of 10mg/ml RNase A was added temporarily), shaken well, 75ul of the second solution (5M NaCl) was added thereto, shaken well, left for 2 minutes, 12000rpm was centrifuged for one minute, the supernatant was taken out and put into a new EP tube, 0.6 times the supernatant volume of isopropanol was added thereto, shaken well, transferred into a centrifugal column, washed 2 times, left for 2 minutes at room temperature, and DNA was eluted from the centrifugal column with 100ul of the eluent. The whole DNA extraction process is completed at room temperature, and takes about 15 minutes.
The results were measured on a biaosharp (table 5) and examined by gel electrophoresis (fig. 5), which shows that the DNA extraction method of the present invention can rapidly extract mouse liver DNA at room temperature.
Table 5: quality of extracted mouse liver tissue DNA
| Animal tissue | Mouse liver |
| DNA concentration | 153.4ng/ul |
| A260/A280 | 1.825 |
The result shows that the method for rapidly extracting the animal tissue DNA provided by the invention has better universality. More remarkably, the operation is carried out at room temperature, and the operation time is very short, so that more possibilities are provided for shortening the whole experiment time and improving the experiment efficiency. All the reagents are nontoxic, and the health and environmental protection of experimenters are facilitated.
Sources of reagents used in the present invention:
although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, combinations, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (11)
1. A green rapid animal tissue DNA extraction method is characterized by comprising the following steps:
(a) treating animal tissue;
(b) transferring the treated animal tissue into one or more solutions;
(c) fixing the animal tissue DNA on a substrate;
(d) washing and eluting the DNA;
2. the method for rapidly extracting DNA from animal tissue in green according to claim 1, wherein the method comprises the following steps: the animal tissue is treated in step (a), and specifically, the animal tissue is changed into fine tissue particles by liquid nitrogen freeze-drying grinding method, mechanical grinding method, enzymolysis method, ultrasonic method and the like. Protective solutions may be used during sample processing, which contain chelating agents and/or buffer compounds, etc.
3. The method for rapidly extracting DNA from animal tissue in green according to claim 1, wherein the method comprises the following steps: the solution of step (b) includes, but is not limited to, 2 or more solutions, wherein the first solution consists of:
-a mixture comprising at least cetylammonium bromide (CTAB) and/or other surfactants, wherein the CTAB concentration is between 1% and 6%;
-at least one or more chelating agent compounds, preferably selected from, including but not limited to: N-acetyl-L-cysteine, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N, N '-disuccinic acid (EDDS), 1, 2-bis (o-aminophenoxy) ethane-N, N' -tetraacetic acid (BAPTA), and phosphonate chelating agents (including, for example and without limitation, nitrilotris (methylene) phosphonic acid (NTMP), ethylenediaminetetra (methylene phosphonic acid) (EDTMP), diethylenetriaminepenta (methylene) phosphonic acid (DTPMP), 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP), etc.), preferably EDTA, at a concentration of 20mM to 300 mM;
-at least one salt solution or a mixture of more than one salt solution, preferably NaCl, in a concentration of 1M to 3M;
-and/or one or more buffer compounds, the pH value of which may be between 3 and 11, preferably selected from, but not limited to: TRIS (hydroxymethyl) aminomethane (TRIS), N- (TRIS (hydroxymethyl) methyl) glycine (TRICINE), N-bis (2-hydroxyethyl) glycine (BICINE), N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid) (HEPES), piperazine-1, 4-bis (2-ethanesulfonic acid) (PIPES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 2- (N-morpholino) ethanesulfonic acid (MES), 3- (N-morpholino) propanesulfonic acid (MOPS) and/or phosphate buffer, sodium acetate buffer, methyl acetate buffer, etc., preferably PH8.0TRIS-HCl, at a concentration of 20mM to 300 mM;
-and/or one or more water-soluble organic substances, preferably selected from, including but not limited to: guanidine hydrochloride, guanidine isothiocyanate, glycerol, glucose, fructose, sucrose, maltose and the like, preferably glycerol, in a concentration of: 1% -10%;
and/or one or more other substances which have little or no effect.
4. The method for extracting DNA from animal tissue according to claim 1, wherein: the second solution in the step (b) is a salt solution and/or a mixture solution of more than one salt, wherein the pH of the salt solution is 2-5M and is 3-11, and sodium chloride with the pH of 7.0 is preferably selected.
5. The method for extracting DNA from animal tissue according to claim 1, wherein: the binding agent used in step (c) includes, but is not limited to, branched or unbranched alkanols, in particular, short chain branched or unbranched alkanols having 1 to 5 carbon atoms. Branched or unbranched butanols including n-butanol, isobutanol, sec-butanol, and tert-butanol; branched or unbranched pentanols include, for example, n-pentanol and isopentyl alcohol. Preference is given to using alcohols selected from the group consisting of: methanol, ethanol, isopropanol, and/or mixtures thereof, it is particularly preferred to use an alcohol selected from the group consisting of: ethanol, isopropanol and/or mixtures thereof, preferably isopropanol, in amounts such that: 0.3-0.9 times of the volume of the supernatant.
6. The method for extracting DNA from animal tissue according to claim 1, wherein: step (c) DNA is immobilized on a spin column or magnetic sphere capable of binding DNA.
7. The method for extracting DNA from animal tissue according to claim 1, wherein: the detergent used in step (d) may be a conventional buffer or any other suitable medium. Generally, detergents having low to moderate ionic strength are preferred, such as 10mM TRIS (hydroxymethyl) aminomethane (TRIS) solutions and/or 0.05M to 0.2M sodium citrate solutions, and the like. A wash buffer having a higher salt concentration, such as a 4-6M solution of guanidine hydrochloride, may also be used. As noted above, the wash reagents of the invention are similarly suitable wash reagents. For example, an aqueous solution of an alcohol having 1 to 5 carbon atoms, preferably an aqueous solution of ethanol, specifically an aqueous solution containing 50 to 100% ethanol. The cleaning times are as follows: once or more than once.
8. The method for extracting DNA from animal tissue according to claim 1, wherein: the elution reagent used in step (d) may be a low salt nucleic acid eluent, such as: the reagent with salt content less than 0.1M is used as the elution reagent with low salt content. Particularly preferred are elution reagents comprising the buffer compound TRIS (hydroxymethyl) aminomethane (TRIS) and/or 0.01% to 0.02% aqueous DEPC solution. Also particularly suitable for elution is demineralized water, optionally containing one or more additives, for example chelating agents such as ethylenediaminetetraacetic acid (EDTA), azide compounds and/or buffer compounds such as TRIS (hydroxymethyl) aminomethane (TRIS) and the like. Elution times: once or more than once.
9. The method for extracting DNA from animal tissue according to claim 1, wherein: steps (b), (c) and (d) are carried out at room temperature, but also at elevated temperature, for example: 30-95 ℃.
10. A kit for performing the method of any one of claims 1 to 8, wherein the kit comprises but is not limited to animal tissue and/or cell green rapid DNA extraction kit, blood green rapid DNA extraction kit, saliva green rapid DNA extraction kit, oral cell green rapid DNA extraction kit, and the like, and a carrier describing the method of any one of claims 1 to 8.
11. A computer readable carrier carrying a computer program comprising instructions for carrying out the method according to any one of claims 1 to 8.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533724A (en) * | 2010-12-30 | 2012-07-04 | 上海复星医学科技发展有限公司 | Cell lysis reagent for extracting and purifying nucleic acids in biological samples |
| CN104232617A (en) * | 2008-05-23 | 2014-12-24 | 生命科技公司 | Methods and kits for extraction of DNA |
| CN104630208A (en) * | 2015-02-11 | 2015-05-20 | 杭州百迈生物技术有限公司 | Kit and method for extracting genome DNA (deoxyribonucleic acid) |
| CN105838708A (en) * | 2016-04-29 | 2016-08-10 | 中国食品发酵工业研究院 | Method for extracting DNA from salmon can |
| CN108342383A (en) * | 2018-02-28 | 2018-07-31 | 默禾医疗科技（上海）有限公司 | A kind of the rapid extraction kit and its extracting method of high-purity DNA or RNA |
| CN111778237A (en) * | 2020-06-30 | 2020-10-16 | 中国测试技术研究院 | High-purity nucleic acid extraction method and reagent system for animal deep-processed products |
| CN111808844A (en) * | 2020-07-29 | 2020-10-23 | 广州捷倍斯生物科技有限公司 | Kit for simultaneously extracting DNA and RNA and use method thereof |
-
2021
- 2021-06-18 CN CN202110707478.8A patent/CN113265399A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104232617A (en) * | 2008-05-23 | 2014-12-24 | 生命科技公司 | Methods and kits for extraction of DNA |
| CN102533724A (en) * | 2010-12-30 | 2012-07-04 | 上海复星医学科技发展有限公司 | Cell lysis reagent for extracting and purifying nucleic acids in biological samples |
| CN104630208A (en) * | 2015-02-11 | 2015-05-20 | 杭州百迈生物技术有限公司 | Kit and method for extracting genome DNA (deoxyribonucleic acid) |
| CN105838708A (en) * | 2016-04-29 | 2016-08-10 | 中国食品发酵工业研究院 | Method for extracting DNA from salmon can |
| CN108342383A (en) * | 2018-02-28 | 2018-07-31 | 默禾医疗科技（上海）有限公司 | A kind of the rapid extraction kit and its extracting method of high-purity DNA or RNA |
| CN111778237A (en) * | 2020-06-30 | 2020-10-16 | 中国测试技术研究院 | High-purity nucleic acid extraction method and reagent system for animal deep-processed products |
| CN111808844A (en) * | 2020-07-29 | 2020-10-23 | 广州捷倍斯生物科技有限公司 | Kit for simultaneously extracting DNA and RNA and use method thereof |
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