CN109797148B - DNA extraction reagent, DNA extraction method and DNA extraction kit - Google Patents
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Abstract
The invention relates to a DNA extraction reagent, a DNA extraction method and a DNA extraction kit. The DNA extraction reagent comprises a lysate and an impurity removing agent, wherein the lysate comprises guanidinium isothiocyanate with the final concentration of 1-5M, Tris-HCl with the final concentration of 0.01-0.2M and N-lauroylsarcosine with the final mass volume percentage of 1-10%; the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone. The lysate and the impurity removing agent in the DNA extraction reagent can be matched with each other, the degradation of DNA in the extraction preparation process can be reduced, the purity of the DNA is improved, and the DNA prepared by using the DNA extraction reagent can meet the requirement of metagenome sequencing.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a DNA extraction reagent, a DNA extraction method and a DNA extraction kit.
Background
Human health is closely related to the number and types of microorganisms on the surface and organs of the human body, and particularly, intestinal microorganisms play an important role in human health. In recent years, a great deal of research has found that various microorganisms in human intestinal tract play an important role in diseases such as immunity, development, inflammation, metabolic diseases, mental disorders, cancer and the like, and research on intestinal microbiota can help us to prevent, diagnose and finally cure or inhibit such diseases.
The fecal sample is one of intestinal microorganism research sources, and the sampling is convenient and noninvasive. However, human feces are complex, fibers, microorganisms, undigested particles, nucleases, human exfoliated cells and the like exist, and it is difficult to remove the fibers and the undigested particles from the feces, thereby affecting the overall quality and quantity of the metagenomic DNA to be analyzed. Furthermore, the presence of these impurities also affects the efficient lysis of the microbial cells. If the lysis of the microorganisms in the sample is insufficient, the high-throughput sequencing result is deviated, and the diversity of the microorganisms in the detected sample is finally influenced. Therefore, extraction of DNA from feces is critical to reveal the diversity of the intestinal flora.
At present, complete genome and high DNA purity are often required when metagenome sequencing research is carried out. However, most of the current commercial extraction kits have the defects of column extraction, the DNA degradation is serious due to the shearing force generated during centrifugal filtration, the DNA yield is low, the metagenome sequencing requirement is difficult to meet, and the cost is high; the DNA obtained by the traditional phenol chloroform extraction method has low purity, and subsequent PCR, high-throughput sequencing and the like are influenced.
Disclosure of Invention
Based on this, it is necessary to provide a DNA extraction reagent capable of reducing DNA degradation and improving DNA purity.
A DNA extraction reagent comprises a lysate and an impurity removing agent, wherein the lysate comprises guanidinium isothiocyanate with the final concentration of 1M-5M, Tris-HCl with the final concentration of 0.01M-0.2M and N-lauroylsarcosine with the final mass volume percentage of 1% -10%; the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone.
The lysate and the impurity removing agent in the DNA extraction reagent can be matched with each other, so that the degradation of DNA in the extraction preparation process can be reduced, the purity of the prepared DNA is improved, and the prepared DNA can meet the requirement of metagenome sequencing.
In addition, a DNA extraction kit and a DNA extraction method capable of reducing DNA degradation and improving DNA purity are also provided.
A DNA extraction kit comprises the DNA extraction reagent.
A method for extracting DNA comprises the following steps:
cracking a sample by using a cracking solution to obtain a solution to be purified, wherein the cracking solution comprises guanidinium isothiocyanate with the final concentration of 1-5M, Tris-HCl with the final concentration of 0.01-0.2M and N-lauroylsarcosine with the final mass volume percentage of 1-10%; and
and treating the solution to be purified by using an impurity removing agent to obtain a crude DNA product, wherein the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone.
Drawings
FIG. 1 is an electrophoretogram of examples 1 and 2;
FIG. 2 is an electrophoretogram of example 3;
FIG. 3 is an electrophoretogram of example 4;
FIG. 4 is an electrophoretogram of example 5.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The invention provides a DNA extraction reagent, which comprises a lysate and an impurity removing agent, wherein the lysate comprises guanidinium isothiocyanate with the final concentration of 1-5M, Tris-HCl with the final concentration of 0.01-0.2M and N-lauroylsarcosine with the final mass volume percentage of 1-10%; the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone.
The lysate and the impurity removing agent in the DNA extraction reagent can be matched with each other, the degradation of DNA in the extraction preparation process can be reduced, the purity of the prepared DNA is improved, and the DNA prepared by using the DNA extraction reagent can meet the requirement of metagenome sequencing.
The embodiment of the invention also provides a DNA extraction kit, which comprises the DNA extraction reagent.
Specifically, the DNA extraction kit further comprises at least one of a TENP buffer, a TE buffer, and a washing solution.
Of course, in some embodiments, the DNA extraction kit may not include TENP buffer, TE buffer, or/and washing solution, and the user may prepare the kit by himself.
The lysate and the impurity removing agent in the DNA extraction kit can be matched with each other, the degradation of DNA in the extraction preparation process can be reduced, the purity of the prepared DNA is improved, and the DNA prepared by using the DNA extraction reagent can meet the requirement of metagenome sequencing.
An embodiment of the present invention further provides a DNA extraction method, including the steps of:
and step S110, preprocessing the sample to obtain a sample to be cracked.
Specifically, the sample is washed with physiological saline or phosphate buffer. Further, mixing the sample with physiological saline with the volume 5-10 times of the sample volume, centrifuging at 1000-5000 rpm/min, taking the supernatant, then centrifuging at 10000-12000 rpm/min, discarding the supernatant, taking the precipitate, then adding PBS for uniformly mixing, centrifuging at 1000-5000 rpm/min, taking the supernatant at 10000-12000 rpm/min, and obtaining the to-be-cracked sample. A part of non-cellular components (such as fibers) in the sample can be removed by washing with physiological saline or phosphate buffer solution and centrifuging at a plurality of different rotation speeds. In this embodiment, the sample is a stool sample.
And S130, cracking the sample by using a cracking solution to obtain a solution to be purified, wherein the cracking solution comprises guanidine isothiocyanate with the final concentration of 1-5M, Tris-HCl with the final concentration of 0.01-0.2M and N-lauroylsarcosine with the final mass volume percentage of 1-10%.
Specifically, mixing a sample with a lysis solution, and then incubating at 56-95 ℃ for 0.5-1.5 hours to obtain an incubation solution; and then grinding the incubation liquid to obtain the liquid to be purified. The volume ratio of the sample to the lysate is 1: 2-8. Preferably, the volume ratio of sample to lysate is 1:4 or 1: 6. further, grinding aids, such as glass beads, are added during grinding to cooperatively grind and accelerate cell lysis.
In one embodiment, the final concentration of guanidinium isothiocyanate in the lysate is from 2M to 4M. Further, the final concentration of guanidinium isothiocyanate in the lysate was 3M or 4M. The final concentration of Tris-HCl in the lysate is 0.05M-0.1M, and the pH of Tris-HCl is 7.5-8.0. Further, the final concentration of Tris-HCl in the lysate was 0.05M, 0.1M. The final mass volume percentage of the N-lauroylsarcosine in the lysate is 2-5%. Further, the final mass volume percentage of N-lauroylsarcosine in the lysate was 3% or 4%.
Further, the lysis solution further comprises a buffer solution, wherein the buffer solution is selected from one of phosphate buffer solution and HEPES buffer solution. Of course, in some embodiments, the lysate may not include a buffer, and the user may mix the buffer with guanidinium isothiocyanate, Tris-HCl, and N-lauroylsarcosine in the DNA extraction reagent after preparing the buffer by himself/herself, as long as the final concentrations of guanidinium isothiocyanate, Tris-HCl, and N-lauroylsarcosine in the lysate after preparation are as described above.
And S150, treating the solution to be purified by using an impurity removing agent to obtain a crude DNA product, wherein the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone.
Specifically, an impurity removing agent is mixed with a solution to be purified, and then centrifugation is performed to obtain a supernatant, thereby obtaining a crude DNA product. The mass ratio of the impurity removing agent to the liquid to be purified is 10 mg-60 mg:1000 muL. Preferably. The mass ratio of the impurity removing agent to the liquid to be purified is 15 mg-30 mg:1000 muL. Furthermore, the volume ratio of the mass of the impurity removing agent to the liquid to be purified is 15mg:1000 muL and 20mg:1000 muL.
Large amounts of intestinal microorganisms, undigested food residues, digestive enzymes, lipid substances, proteins, RNA, polysaccharides, plant secondary metabolites, etc. are present in human feces. PVPP (crosslinked polyvinylpyrrolidone) and PVP (polyvinylpyrrolidone) can reduce the influence of phenols, quinones and tannins on DNA purity. PVPP and PVP can complex polyphenol substances, effectively prevent the polyphenol substances from being oxidized into quinones and avoid the solution from being browned.
In one embodiment, after the steps of mixing the decontaminating agent with the liquid to be purified, centrifuging, and taking the supernatant, the method further comprises the step of washing the precipitate after centrifuging. The recovery rate of DNA can be improved by washing the precipitate by centrifugation. Specifically, the pellet after centrifugation was washed with TENP buffer, followed by centrifugation, and the supernatants were combined to obtain a crude DNA product. The TENP buffer solution comprises Tris (pH8.0) with the final concentration of 10 mM-100 mM, EDTA (pH8.0) with the final concentration of 10 mM-50 mM, NaCl with the final concentration of 50 mM-500 mM and PVPP with the final mass volume percentage of 1% -6%. Further, the TENP buffer solution comprises Tris (pH8.0) with the final concentration of 20 mM-50 mM, EDTA (pH8.0) with the final concentration of 10 mM-20 mM, NaCl with the final concentration of 100 mM-200 mM and PVPP with the final mass volume percentage of 1% -6%.
In one embodiment, the dedoping agent comprises crosslinked polyvinylpyrrolidone or polyvinylpyrrolidone.
And step S170, treating the crude DNA product with a washing solution to obtain purified DNA.
Specifically, the washing solution includes a first washing solution and a second washing solution. The first washing solution comprises phosphate with a final concentration of 0.01-0.2M and potassium acetate with a final concentration of 0.5-2M. The second wash solution includes sodium acetate at a final concentration of 3M to 5M. The first wash solution aids in the removal of proteins. The second wash solution helps to precipitate the DNA, allowing the DNA to be separated from other impurities. Further, the first washing solution comprises phosphate with a final concentration of 0.05M to 0.1M and potassium acetate with a final concentration of 1M to 1.5M. Preferably, the first wash solution comprises phosphate at a final concentration of 0.05M or 0.1M, and potassium acetate at a final concentration of 1M or 1.5M. The second wash solution included sodium acetate at a final concentration of 3M or 4M.
In one embodiment, isopropanol is mixed with the crude DNA product and the DNA is pelleted by centrifugation to remove protein. Then, the precipitate containing the DNA is mixed with the first washing solution, and then centrifuged again to further precipitate the protein to remove the protein in the DNA. Then, the supernatant is taken out and added with a second washing solution and absolute ethyl alcohol, and the mixture is placed at the temperature of minus 20 ℃ for more than 8 hours to promote the DNA precipitation so as to remove impurities such as protein in the DNA. Then, cations in the DNA were removed with 70% ethanol to obtain purified DNA. Further, the method further comprises a step of adding RNase during the step of washing the DNA with the first detergent and the second detergent.
The extraction method of the DNA is simple and convenient to operate, has low extraction cost relative to a kit, can reduce the degradation of the DNA, has high purity of the obtained DNA, and can meet the requirement of metagenome sequencing.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples, unless otherwise specified, do not include other components except for inevitable impurities. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.
The stool samples of examples 1 to 5 were all from the same batch of stools of the same person.
Example 1
Three stool samples from the same person, 200mg each, were weighed separately and the following operations were performed for each stool sample:
(1) pretreatment: mixing the fecal sample with 5 times volume of physiological saline, vortex oscillating, mixing, centrifuging at 4 deg.C at 2000rpm/min for 2min, collecting supernatant, discarding precipitate, centrifuging at 4 deg.C at 10000rpm/min for 1min, discarding supernatant, and retaining the precipitate after centrifugation. Adding 1mL of PBS (pH 7.4) into the precipitate, vortexing, shaking and mixing uniformly, centrifuging at the temperature of 4 ℃ at the speed of 2000rpm/min for 2min, taking the centrifuged supernatant, and removing the precipitate; and centrifuging the centrifuged supernatant for 1min at the speed of 10000rpm/min at the temperature of 4 ℃, discarding the supernatant, and reserving the precipitate, wherein the precipitate is purified microbial cells and is a sample to be lysed.
(2) Cracking a sample by using a cracking solution: adding 800 mu L of lysis solution into the sample to be lysed obtained in the step (1), vortexing, shaking and mixing uniformly, and incubating for 1 hour at 70 ℃. Wherein the lysis solution comprises guanidinium isothiocyanate with a final concentration of 4M, Tris-HCl with a final concentration of 0.1M, N-lauroylsarcosine with a final mass-volume percentage of 3%, and phosphate buffer solution with a final concentration of 0.1M and a pH value of 8.0. After incubation, 750mg of 0.1mm glass beads were added, vortexed, and then cryoground at-30 ℃ for 5 minutes, suspended for 10 minutes, and further ground for 5 minutes to obtain a solution to be purified.
(3) Treating the solution to be purified with an impurity removing agent: adding 15mg of PVPP into the solution to be purified obtained in the step (2), uniformly mixing by vortex oscillation, centrifuging for 5min at the temperature of 4 ℃ and the rpm of 14000, taking out the supernatant, placing the supernatant into a new 2mL centrifuge tube, and putting the centrifuge tube on ice. Then, 500. mu.L of TENP buffer was added to the centrifuged pellet, vortexed, mixed well, centrifuged at 14000rpm at 4 ℃ for 5min, and the supernatant was carefully aspirated and combined with the supernatant obtained in the previous operation. The TENP buffer consisted of Tris pH8.0 at a final concentration of 50mM, EDTA pH8.0 at a final concentration of 20mM, NaCl at a final concentration of 100mM and PVPP at 1% final mass volume. The centrifuged pellet was washed twice with TENP buffer and the supernatants were combined after each centrifugation to give crude DNA.
(4) Further purification of the crude DNA: the DNA crude product obtained in step (3) is evenly distributed into 2mL centrifuge tubes, and each centrifuge tube is subjected to the following operations:
adding 1mL of isopropanol into the centrifuge tube, reversing and uniformly mixing, standing at 25 ℃ for 15 minutes, centrifuging at 14000rpm at 4 ℃ for 10min, removing supernatant, and inversely placing the centrifuge tube containing the precipitate on a paper towel for drying. Then 500. mu.L of the first wash was added to dissolve the precipitate sufficiently, and the precipitate was incubated on ice for 90 minutes. Wherein the first washing solution is composed of phosphate buffer solution with final concentration of 0.1M and pH8.0, and potassium acetate with final concentration of 1M. Incubating on ice for 90 min, and centrifuging at 14000rpm at 4 deg.C for 30 min; the supernatant was removed and 2. mu.L RNase (10mg/mL) was added, vortexed and incubated at 37 ℃ for 30 min. After incubation at 37 ℃ for 30min, 50. mu.L of a second wash solution of 3M sodium acetate at pH 5.2 and 1mL of fresh absolute ethanol (stored at-20 ℃) were added, mixed by inversion and left overnight at-20 ℃. Then centrifuged at 14000rpm for 30min at 4 ℃ and the ethanol was discarded. Then 1mL of 70% ethanol was added, the centrifuge tube was flicked several times to suspend the DNA in 70% ethanol, and then centrifuged at 14000rpm for 5min, and 70% ethanol was discarded. Then, 500. mu.L of 70% ethanol was added, the centrifugal tube was flicked several times to make the DNA re-suspended in 70% ethanol, and the DNA was centrifuged at 14000rpm for 5min, and 70% ethanol was discarded to obtain purified DNA. To the purified DNA, 100. mu.L of buffer TE consisting of Tris-HCl at a final concentration of 10mM, pH8.0 and EDTA at a final concentration of 1mM, pH8.0 was added and stored for later use.
(5) The three groups of DNA prepared in example 1 were checked for purity by Nano drop, the three groups of DNA prepared in example 1 were checked for concentration by Qubit3.0, and the integrity of the DNA was checked by electrophoresis separately. The purity and concentration of DNA in the three fecal samples of example 1 are shown in Table 1 and the electrophoretogram is shown in FIG. 1. In Table 1, the numbers 2 to 4 represent the extraction results of three stool samples of example 1. Number 1 in fig. 1 corresponds to e.coli DNA standard, numbers 2-4 correspond to electrophoresis results of three stool samples of example 1; m corresponds to 15K DNA ladder.
Example 2
Example 2 the procedure and method for extracting DNA from feces was the same as in example 1, except that the lysis solution used in example 2 consisted of guanidine isothiocyanate at a final concentration of 1M, Tris-HCl at a final concentration of 0.05M, N-lauroylsarcosine at a final mass-volume percentage of 5%, and phosphate buffer at a final concentration of 0.05M and pH 8.0. The TENP buffer consisted of 100mM Tris pH8.0 final concentration, 50mM EDTA pH8.0 final concentration, 500mM NaCl final concentration and 6% PVPP final mass volume percent. The first wash consisted of phosphate at a final concentration of 0.05M pH8.0 and potassium acetate at a final concentration of 1.5M.
The concentrations of DNA in the three fecal samples of example 2 are shown in Table 1, and the electrophoretogram is shown in FIG. 1. In Table 1, the numbers 5 to 7 represent the extraction results of three stool samples of example 2. Coli DNA standard in fig. 1, and 5-7 in the figure, which correspond to the electrophoresis results of the three stool samples of example 2. As is clear from FIG. 1, the DNAs obtained in example 1 and example 2 were not degraded.
TABLE 1
Example 3
Example 3 DNA was extracted from feces according to the conventional SDS manual method, specifically:
200mg of human excrement sample is put into a 2mL centrifuge tube, 500 mu L of SDS lysate with the final mass-volume ratio of 2% is added, and the mixture is shaken and mixed evenly. The 2% SDS lysate consisted of a final concentration of 500mM NaCl,50mM Tris-HCl (pH 8),50mM EDTA and a final percentage by mass of 2% SDS. Then, 500. mu.L of TE-saturated phenol was added thereto, and the mixture was mixed by turning upside down, and then 0.3g of grinding beads were added thereto and the mixture was ground in a grinder for 30 seconds. The milled samples were centrifuged at 15000rpm for 5min at 4 ℃, the centrifuged supernatant was pipetted into a 2mL fresh centrifuge tube and 400 μ L final volume ratio of 25: 24: 1, and grinding the centrifugal tube in a grinder for 45 seconds. Centrifuging the ground sample at 4 ℃ and 15000rpm for 5min, sucking the supernatant into a new centrifuge tube, adding equal volume of isopropanol and 1/10 volume of 3M sodium acetate, turning the centrifuge tube upside down and mixing uniformly, centrifuging at 4 ℃ and 15000rpm for 5min, and discarding the supernatant. Drying the DNA precipitate in a clean bench for 10min, adding 50. mu.L TE buffer solution to dissolve the DNA, storing at 4 ℃ overnight, taking out, adding 2. mu.L RNase (10mg/mL), heating at 37 ℃ for 30min, and storing at-20 ℃ for a long time.
The results of example 3 are shown in table 2 and fig. 2. Numbers 1-3 in Table 1 correspond to three stool samples of example 3. In FIG. 2, M is a 15K DNA ladder, and the numbers 1 to 3 correspond to the electrophoresis results of the numbers 1 to 3 in Table 2. As is clear from FIG. 2, the DNA obtained in example 3 was partially degraded.
TABLE 2
| Numbering | Concentration (ng/. mu.L) | Total amount of μ g | OD260/ |
| 1 | 146 | 7.3 | 1.90 |
| 2 | 77.8 | 3.89 | 1.91 |
| 3 | 125 | 6.25 | 1.90 |
Example 4
Example 4 DNA in feces was extracted using QIAamp Fast DNA pool Mini kit and Tiangen feces kit, respectively, and the detailed procedures were performed according to the instructions of the kits.
The results of example 4 are shown in table 3 and fig. 3. In Table 3, the numbers 1-3 are the extraction results of the QIAamp Fast DNA Stool Mini kit, and the numbers 4-5 are the extraction results of the Tiangen feces kit. The numbers 1-3 in FIG. 3 correspond to the electrophoresis results of the QIAamp Fast DNA pool Mini kit; the numbers 4-5 correspond to electrophoresis results of the Tiangen fecal kit; code 6 corresponds to e.coli DNA standard; m corresponds to 15K DNA ladder. As can be seen from FIG. 3, the DNA extracted by the two kits of example 4 was severely degraded.
TABLE 3
| Numbering | Concentration (ng/. mu.L) | Total amount of μ g | OD260/ |
| 1 | 31.6 | 1.52 | 1.76 |
| 2 | 35.6 | 1.78 | 1.74 |
| 3 | 37.6 | 1.88 | 1.78 |
| 4 | 30.2 | 1.51 | 1.68 |
| 5 | 28.2 | 1.41 | 1.73 |
Example 5
Example 5 the procedure and method for extracting DNA from feces were substantially the same as in example 1, except that step (3) in example 1 was omitted.
The results of example 5 are shown in Table 4 and FIG. 4, and it can be seen that the DNA purity obtained by extraction is low in Table 4 in which number 1 corresponds to the extraction result of example 5. When OD260/OD 280. apprxeq.1.8, the DNA was considered to be relatively pure, and the total amount of DNA obtained without recovery of the cleaved DNA with the TENP buffer was lower than in example 1. In FIG. 4, M corresponds to 15K DNA ladder, and number 1 corresponds to the electrophoresis result of example 5. As can be seen from FIG. 4, the DNA of example 5 was also partially degraded.
TABLE 4
| Numbering | Concentration (ng/. mu.L) | Total amount of μ g | OD260/ |
| 1 | 57.6 | 3.46 | 1.69 |
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A DNA extraction reagent is characterized by comprising a lysate and an impurity removal agent, wherein the lysate comprises guanidinium isothiocyanate with the final concentration of 1-5M, Tris-HCl with the final concentration of 0.01-0.2M, N-lauroylsarcosine with the final mass volume percentage of 1-10% and a phosphate buffer solution; the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone.
2. The DNA extraction reagent according to claim 1, wherein the final concentration of guanidinium isothiocyanate in the lysate is 2M to 4M; and/or the final concentration of Tris-HCl in the lysate is 0.05M-0.1M; and/or the final mass volume percentage of the N-lauroylsarcosine in the lysate is 2-5%.
3. A DNA extraction kit comprising the DNA extraction reagent according to claim 1 or 2.
4. The DNA extraction kit according to claim 3, further comprising at least one of a TENP buffer, a TE buffer and a washing solution.
5. The DNA extraction kit according to claim 4, wherein the washing solution comprises a first washing solution and a second washing solution, and the first washing solution comprises a phosphate and a potassium acetate at final concentrations of 0.01-0.5M and 0.5-2M; the second washing solution comprises sodium acetate with the final concentration of 3M-5M.
6. A method for extracting DNA, which is characterized by comprising the following steps:
cracking a sample by using a cracking solution to obtain a solution to be purified, wherein the cracking solution consists of guanidinium isothiocyanate with the final concentration of 1-5M, Tris-HCl with the final concentration of 0.01-0.2M, N-lauroylsarcosine with the final mass volume percentage of 1-10% and a phosphate buffer solution, and the sample is a microbial cell; and
and treating the solution to be purified by using an impurity removing agent to obtain a crude DNA product, wherein the impurity removing agent comprises at least one of cross-linked polyvinylpyrrolidone and polyvinylpyrrolidone.
7. The method for extracting DNA according to claim 6, wherein in the step of lysing a sample with a lysate, the volume ratio of the sample to the lysate is 1:2 to 8.
8. The method for extracting DNA according to claim 6, wherein the step of lysing the sample with the lysis solution to obtain the solution to be purified comprises:
mixing the sample with the lysate, and incubating at 56-95 ℃ to obtain an incubation solution; and
and grinding the incubation liquid to obtain the liquid to be purified.
9. The method for extracting DNA according to any one of claims 6 to 8, further comprising a step of washing the sample with physiological saline or a phosphate buffer solution to pretreat the sample before the step of lysing the sample with the lysis solution.
10. The method for extracting DNA according to any one of claims 6 to 8, further comprising a step of treating the crude DNA product with a first washing solution and a second washing solution to obtain purified DNA after the step of treating the solution to be purified with an impurity removing agent to obtain a crude DNA product; wherein the first washing solution comprises phosphate with the final concentration of 0.01-0.5M and 0.5-2M potassium acetate; the second washing solution comprises sodium acetate with the final concentration of 3M-5M.
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Families Citing this family (2)
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| CN111534466B (en) * | 2020-05-14 | 2021-06-04 | 深圳市融思生物科技有限公司 | Method and reagent for processing human fecal intestinal flora sample |
| CN112195177B (en) * | 2020-10-28 | 2021-08-06 | 上海慕柏生物医学科技有限公司 | Nucleic acid extraction method and kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684137A (en) * | 2008-09-26 | 2010-03-31 | 贵州仁怀茅台镇金士酒业有限公司 | Method for extracting total DNA of microorganism in liquor Daqu |
-
2019
- 2019-02-02 CN CN201910107329.0A patent/CN109797148B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684137A (en) * | 2008-09-26 | 2010-03-31 | 贵州仁怀茅台镇金士酒业有限公司 | Method for extracting total DNA of microorganism in liquor Daqu |
Non-Patent Citations (3)
| Title |
|---|
| 几种真菌DNA提取方法的比较;吴发红等;《中国农学通报》;20090605;第25卷(第8期);第62-64页 * |
| 大黄牡丹汤对肠道菌群的体外作用;郑彦懿等;《广州中医药大学学报》;20160613;第33卷(第3期);第357-361页 * |
| 郑彦懿等.大黄牡丹汤对肠道菌群的体外作用.《广州中医药大学学报》.2016,第33卷(第3期),第357-361页. * |
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| KR20230060850A (en) * | 2021-10-28 | 2023-05-08 | (주)한국첨단시험연구원 | Precipitant Composition for DNA extraction, and Method for DNA Extraction thereby |
| KR102603588B1 (en) | 2021-10-28 | 2023-11-20 | (주)한국첨단시험연구원 | Precipitant Composition for DNA extraction, and Method for DNA Extraction thereby |
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