Sample processing method for rapidly extracting biological nucleic acid
阅读说明：本技术 一种快速提取生物核酸的样品处理方法 (Sample processing method for rapidly extracting biological nucleic acid ) 是由 张怀远 丁玮云 李海文 于 2021-08-11 设计创作，主要内容包括：本发明提供一种快速提取生物核酸的样品处理方法——浓缩酶解法、生物核酸提取方法以及试剂盒。该方法具有下列优点：1、操作简单,细胞酶解更彻底；2、极大缩短提取时间,提高工作效率；3、提高生物核酸的产量；4、室温。(The invention provides a sample processing method for quickly extracting biological nucleic acid, namely a concentration enzymolysis method, a biological nucleic acid extraction method and a kit. The method has the following advantages: 1. the operation is simple, and the cell enzymolysis is more thorough; 2. the extraction time is greatly shortened, and the working efficiency is improved; 3. increasing the yield of biological nucleic acid; 4. and (4) room temperature.)
1. A sample processing method for rapidly extracting biological nucleic acid is characterized by comprising the following steps:
a) treating a biological sample, including but not limited to liquid nitrogen cryo-milling, mechanical milling, high speed centrifugation, and/or addition of inhibitors to prevent nucleic acid degradation, etc., to obtain small volumes of cells and/or very small biological tissue aggregates;
b) proteases including, but not limited to, proteinase K, proteases, zymozymes, achromopeptidases, cytolytic enzymes, lysostaphins, lysozymes, ribozymes, and the like are added in small volumes at high concentrations.
2. The method according to claim 1a), wherein: adding and/or nucleic acid degradation inhibitor and/or high-concentration enzyme into a solid biological sample, and obtaining superfine biological sample tissues by a liquid nitrogen freezing grinding method, a mechanical grinding method and the like; for a biological sample containing cells in a liquid, and/or an inhibitor for preventing degradation of nucleic acids is added, and the supernatant is centrifuged to obtain cells of the biological sample.
3. The method according to claim 1b), wherein: and/or adding high concentration protease, specifically more than 0.2mg/ml, preferably 2 mg/ml-50 mg/ml, more preferably 5 mg/ml-30 mg/ml; the volume of the used high-concentration protease is 1 ul-100 ul, preferably 5 ul-50 ul, more preferably 10 ul-30 ul; mixing the enzyme and the biological sample treated in step a) and standing for a period of time, preferably 1-10 minutes. More preferably 1 minute to 3 minutes.
4. According to an embodiment of claim 1, the method for extracting nucleic acid from a biological sample after treatment comprises the following steps:
A) uniformly mixing the biological sample after enzymolysis with a lysate;
B) immobilizing the released nucleic acids onto a substrate or a mixture of more than one substrate, including but not limited to spin columns, magnetic spheres, etc., in the presence of a lysis solution and/or solution containing nucleic acids and/or branched or unbranched alkanols;
C) optionally washing the nucleic acid immobilized on the matrix;
D) optionally eluting the nucleic acid bound to the matrix.
5. The method of claim 4B), wherein the binding agent comprises, 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.
6. The process according to claim 4C), wherein the detergent used is 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. Wash buffers with higher salt concentrations, such as 4M to 6M guanidine hydrochloride solutions, 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.
7. The method according to claim 4D), wherein the elution reagent is a nucleic acid eluent with low salt content, such as: the reagent with salt content less than 0.1M is used as the elution reagent with low salt content. Particularly preferred are an elution reagent containing a buffer compound TRIS (hydroxymethyl) aminomethane (TRIS) and/or an aqueous solution of 0.01% to 0.02% DEPC. 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. It is also possible to use a solution containing 0.2% DEPC water treated at high temperature and high pressure. Elution times: once or more than once.
8. According to claims 1b) and 4D), characterized in that: step 1B) and step 4 (a), (B), (C) and (D) are all performed at room temperature, but may also be performed at elevated temperature, for example: 30-95 ℃.
9. A kit for performing the method according to any one of claims 1 to 9, specifically, but not limited to, a rapid extraction kit for salivary nucleic acid, a rapid extraction kit for oral cellular nucleic acid, an animal tissue and cellular nucleic acid, and the like, and a vector describing the method according to any one of claims 1 to 9.
10. A computer readable carrier carrying a computer program comprising instructions for carrying out the method according to any one of claims 1 to 9. A connected controller; the controller comprises the computer readable carrier of claim 9.
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of biological engineering, and particularly relates to a sample processing method for quickly extracting biological nucleic acid, a nucleic acid extraction method 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. With the development of biology, researchers are gaining favor in the research of RNA, a transcription product of a gene.
More and more extraction methods of DNA, RNA and both are being developed. Specifically, the conventional methods for extracting biological genomic DNA mainly include CTAB and SDS methods, and the common methods for extracting biological RNA mainly include Trizol method, guanidine thiocyanate/phenol method, SDS/phenol method, guanidine hydrochloride method, and the like.
The quality and quantity of nucleic acid extraction for a biological sample are not only related to the extraction recipe but also to the way and/or method of processing the biological sample. Thus, some methods for processing biological samples are emerging continuously, such as: mechanical methods, including high speed tissue mashers, glass homogenizers, and the like; physical methods including repeated freeze thawing, cold and hot alternation, ultrasonic method, and pressure crushing method; chemical and biological methods including organic solvent extraction, aqueous solution extraction, enzymatic methods, and the like.
Among the means and methods widely used in laboratories are mainly mechanical methods, enzymatic methods and liquid nitrogen milling methods. Each of these methods has its advantages and disadvantages. The mechanical method has the advantages that: the efficiency is high, and the automation is facilitated; the disadvantages are as follows: the cost is high. An enzyme method comprises the following steps: the liquid nitrogen freeze drying grinding method has the advantages that: good grinding effect, can quickly extract biological nucleic acid, and has the following defects: is not conducive to automation. The traditional enzyme method has the advantages that: is beneficial to automation and has the following defects: the enzymolysis speed is slow.
[ summary of the invention ]
It is an object of the present invention to provide a method and a room temperature rapid nucleic acid extraction kit that overcome at least one of the above-mentioned drawbacks of the prior art.
The method as claimed in claim 1, wherein the method comprises the following steps:
a) treating a biological sample, including but not limited to liquid nitrogen cryo-milling, mechanical milling, high speed centrifugation, and/or addition of inhibitors to prevent nucleic acid degradation, etc., to obtain small volumes of cells and/or very small biological tissue aggregates;
b) adding a small volume of a high concentration of a protease, including, but not limited to, proteinase K, lysozyme, trypsin, chymotrypsin, etc.;
according to claim 1a), characterized in that: adding and/or nucleic acid degradation inhibitor and/or high-concentration enzyme into a solid biological sample, and obtaining superfine biological sample tissues by a liquid nitrogen freezing grinding method, a mechanical grinding method and the like; for a biological sample containing cells in a liquid, and/or an inhibitor for preventing degradation of nucleic acids is added, and the supernatant is centrifuged to obtain cells of the biological sample.
In order to distinguish from the traditional enzymolysis method, the enzymolysis method provided by the patent is characterized by small volume and high concentration of enzyme, so the method is called a concentrated enzymolysis method.
More specifically, for DNA degradation inhibitors including, but not limited to, and/or 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 (methylenephosphonic acid) (EDTMP), diethylenetriaminepenta (methylene) phosphonic acid (DTPMP), 1-hydroxyethylidene-1, 1-diphosphonic acid (HEDP), and the like), preferably EDTA.
More specifically, for inhibitors of RNA degradation, but not limited to, some reducing agents disclosed in U.S. patent No. 6825340 specification or U.S. patent No. 677720 specification are included. In some embodiments, the commonly used RNaseA inhibitor is an aqueous solution of Diethylpyrocarbonate (DEPC).
In a preferred embodiment, after the high-concentration enzyme is uniformly mixed with the cells and/or the ultrafine biological sample tissues, the total volume is still very small, so that the action distance between the enzyme and the substrate is shortened to accelerate the enzymolysis reaction, the cells and/or the ultrafine biological sample tissues are completely enzymolyzed, the biological nucleic acid is exposed and fully contacted with the nucleic acid extracting solution, the nucleic acid extracting process can be accelerated, and the nucleic acid extracting amount can be increased.
The method according to claim 1b), wherein: and/or adding high concentration protease, specifically more than 0.5mg/ml, preferably 2 mg/ml-50 mg/ml, more preferably 5 mg/ml-30 mg/ml; the volume of the used high-concentration protease is 1 ul-100 ul, preferably 5 ul-50 ul, more preferably 10 ul-30 ul; mixing the enzyme and the biological sample treated in step a) and standing for a period of time, preferably 1-10 minutes. More preferably 1 minute to 3 minutes.
In order to distinguish from the conventional enzymatic hydrolysis method, the enzymatic hydrolysis method provided by the patent is characterized by small volume and high concentration of enzyme, so the method is defined as a concentrated enzymatic hydrolysis method.
For the purposes of the present invention, the term "nucleic acid" is meant to include, but is not limited to: naturally, preferably isolated, linear, branched or cyclic nucleic acids such as RNA, in particular mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA, microRNA or ribozymes, DNA, plasmid DNA, mitochondrial DNA 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.
In a preferred embodiment, the method for extracting nucleic acid from a biological sample after treatment comprises the following steps:
A) uniformly mixing the biological sample after enzymolysis with a lysate;
B) immobilizing the released nucleic acids onto a substrate or a mixture of more than one substrate, including but not limited to spin columns, magnetic spheres, etc., in the presence of a lysis solution and/or solution containing nucleic acids and/or branched or unbranched alkanols;
C) optionally washing the nucleic acid immobilized on the matrix;
D) optionally eluting the nucleic acid bound to the matrix.
In a preferred embodiment, especially the lysis solution may further comprise and/or temporarily add enzymes such as lyase, RNase a, DNase I, in particular, e.g. proteinase K, protease, zymolase, achromopeptidase, cytolytic enzyme, lysostaphin, lysozyme and ribozymes, etc.
The nucleic acids are immobilized on the matrix in the presence of a lysis solution, preferably in the presence of a branched or unbranched alkanol. 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 alkanols", in particular ethanol, propanol, butanol and pentanol, includes the isomeric forms of any of the particular groups which can 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 of said method, said buffer compound is selected from: 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, the lysis and/or binding composition comprises at least one buffer compound selected from the group consisting of: 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.
Biological samples can be lysed at room temperature, e.g., between 15 ℃ and 25 ℃, or at elevated temperatures, e.g., between 30 ℃ and 95 ℃.
In a preferred embodiment, the lysate or lysate mixture may further comprise enzymes, such as proteinase K, protease, lysozyme, staphylococci etc., and depending on the application, nucleases, such as DNase and/or RNAse.
The nucleic acids are immobilized to the spin column in the presence of a salt solution, preferably a branched or unbranched alkanol. 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.
According to a preferred embodiment, the bonding composition comprises a content by volume ranging from 10% to 200%, preferably from 40% to 120%, more preferably from 50% to 80%, based on the total volume of the bonding composition.
Unless otherwise indicated, the definition "branched or unbranched alkanol", in particular ethanol, 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 of the invention, the nucleic acids are isolated by contacting the sample with a matrix based on one or more silica compounds, such as biological sample nucleic acids, and silica, silicate, 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. Incubation times from 10 seconds to 30 minutes are convenient for nucleic acids. According to the method described above, a particularly preferred embodiment, an incubation time of from 1 minute to 10 minutes is convenient for the nucleic acid. According to the above method, according to yet a more preferred embodiment, an incubation time of about 5 minutes is advantageous.
Magnetic particles with a coating of colloidal silica are preferred for isolating nucleic acids. The nucleic acids are 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 having a colloidal silica coating and an average particle size between 6um and 10um, preferably a narrow particle size distribution, are used to isolate nucleic acids.
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, mixtures based on one matrix or on a plurality of silica compound matrices in the form of magnetically or magnetically attractable particles having a silica surface can be used.
The binding of the nucleic acids to the matrix is carried out at a temperature between 4 ℃ and 95 ℃, 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 nucleic acid immobilized on the substrate may be washed prior to removal. Preferably involving resuspension of the particles, for example 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/or a 0.05M to 0.2M sodium citrate solution, and the like. Wash buffers with higher salt concentrations, such as 4M to 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 reagent, 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 nucleic acid 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 nucleic acids are washed again with an aqueous solution containing 70% to 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.
According to step D) of the method, the bound nucleic acids may be removed from the matrix. The process of removing nucleic acids is called elution.
It is also preferred to use nucleic acids bound to a matrix, in particular magnetic particles, without the need for removal steps, for example for PCR or other amplification methods, DNA detection methods or DNA identification methods.
The bound nucleic acids 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. Particularly preferred are an elution reagent containing a buffer compound TRIS (hydroxymethyl) aminomethane (TRIS) and/or an aqueous solution of 0.01% to 0.02% DEPC. 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 particular, the use of the lysis and/or binding composition results in a particularly advantageous method for isolating nucleic acids from biological samples, in particular for isolating biological nucleic acids.
The advantage of the lysis and/or binding agent, even after storage, is, inter alia, that a good product is obtained.
The invention also relates to a kit for isolating and/or purifying nucleic acids from a biological sample containing nucleic acids, which kit comprises the lysis, binding and/or washing reagents of the invention.
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 10mM TRIS (hydroxymethyl) aminomethane (TRIS) solution and/or EDTA and/or 0.05M to 0.2M sodium citrate solution, and the like. Wash buffers with higher salt concentrations, such as 4M to 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 reagent, 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 nucleic acid immobilized to the substrate is preferably washed several times, e.g.2 to 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 nucleic acids are washed again with an aqueous solution containing 70% to 100% ethanol.
According to such embodiments, the bound nucleic acids may be removed from the matrix. The process of removing nucleic acids is called elution of nucleic acids. It is also preferred to use nucleic acids bound to a substrate, in particular magnetic particles, without removal steps, for example for PCR or other amplification methods, DNA detection methods or DNA identification methods, etc.
The bound nucleic acids 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. 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).
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 the lysis, binding, washing and/or elution reagents described above is typically a paper insert, but can be any carrier for an electronic version of the method (including but not limited to removable disks, optical disks, electronic ink screens, network resources and addresses thereof) as long as the method is known by reading the carrier, and is within the scope of the present 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 processing method for rapidly extracting the biological nucleic acid sample provided by the invention at least has the following beneficial effects:
1. the operation is simple, the time is saved, and the extraction of the biological nucleic acid is about 15 minutes;
2. the extraction amount of nucleic acid of the biological sample is increased, and the biological sample is more thoroughly enzymolyzed due to high-concentration enzyme;
3. both the biological sample treatment and the nucleic acid extraction are performed at room temperature, but may be performed by incubation at a high temperature.
[ description of the drawings ]
FIG. 1 is a comparison of a salivary cell before and after enzymatic digestion;
FIG. 2 is a graph of DNA extraction versus gel electrophoresis of saliva cells treated by the enzymatic hydrolysis method of the present invention and liquid nitrogen freeze-drying method, respectively;
FIG. 3 is a graph of DNA extraction versus gel electrophoresis for oral cells treated by the enzymatic hydrolysis method of the present invention and liquid nitrogen freeze-drying method, respectively;
[ 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 1-2 different treatment methods for extraction of DNA from salivary cells
As described above, 800ul of fresh saliva was added to 36ul of 0.5M EDTA pH 8.0, mixed well, divided into two portions, centrifuged at 12000rpm for 1 minute, and the supernatant removed. Treating the first part with liquid nitrogen freeze drying grinding method to obtain saliva cell; adding 10ul of protease K20 mg/ml into the second part, mixing uniformly, standing at room temperature for 2 minutes for enzymolysis (see figure 1), then respectively transferring into 300ul of first lysate (temporarily adding 5ul of RNase A10 mg/ml, green rapid saliva DNA extraction kit), shaking uniformly, then adding 45ul of second lysate (green rapid saliva DNA extraction kit), shaking uniformly, standing for 2 minutes at 12000rpm, centrifuging for 1 minute, taking the supernatant into a new EP tube, adding isopropanol with the volume 0.6 times of the supernatant, shaking uniformly, transferring into a centrifugal column, washing for 2 times, standing at room temperature for 2 minutes, and eluting DNA from the centrifugal column by using 60ul of eluent. The whole DNA extraction process is completed at room temperature, and takes about 15 minutes.
The results were measured on a biaosharp (table 1) and examined by gel electrophoresis (fig. 2), and it can be seen that the present invention provides a concentrated enzymatic method that allows more complete enzymatic digestion of cells and greater amounts of salivary cell DNA extraction than liquid nitrogen freeze-drying.
Table 1: 2 different treatment methods for extracting DNA quality of salivary cells
Processing method
Enzymolysis method
Grinding method
DNA concentration
82.38ng/ul
56.27ng/ul
A260/A280
1.829
1.836
Example 2 extraction of oral cell DNA by 2 different treatment methods
As described above, buccal cells were collected by using buccal swab sticks, and 1ml of a protective solution (10mM Tris-HCl, 20mM EDTA) was added thereto, followed by vigorous shaking, mixing and homogenizing the buccal cells of 2 mouthpieces, centrifuging at 12000rpm for 1 minute, and removing the supernatant. Treating oral cells with liquid nitrogen freeze-drying grinding method; adding 10ul of protease K with the concentration of 20mg/ml into the second part, mixing uniformly, and standing for 2 minutes at room temperature for enzymolysis. Then, 300ul of the first lysate (5 ul of 10mg/ml RNase A, green rapid oral swab DNA extraction kit was added temporarily) was transferred to each other, shaken up, 80ul of the second lysate (green rapid oral swab DNA extraction kit) was added, shaken up, left for 3 minutes at 12000rpm, centrifuged for one minute, the supernatant was taken out and put into a new EP tube, 0.6 times the volume of the supernatant in isopropanol was added, shaken up, transferred to a centrifugal column, washed 2 times, left at room temperature for 2 minutes, and the DNA was eluted from the centrifugal 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 2) and examined by gel electrophoresis (fig. 3), which provides a concentrated enzymatic method that extracts more DNA from buccal cells than by liquid nitrogen freeze-drying.
Table 2: 2 different treatment methods for extracting DNA quality of oral cells
Processing method
Enzymolysis method
Grinding method
DNA concentration
95.68ng/ul
62.31ng/ul
A260/A280
1.835
1.827
The results show that the concentration enzymolysis method provided by the invention can lead the enzymolysis of the biological sample to be more thorough and has better universality in the aspect of the enzymolysis of the biological tissues. 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.
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.