CN114410621A - Simple, convenient and rapid high-throughput genome naked DNA extraction method - Google Patents
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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
Abstract
The invention discloses a simple, convenient and rapid method for extracting naked DNA of a high-throughput genome, which can be used for extracting the genomic DNA of a mammalian cell. The method has high sensitivity, has extremely low requirement on the initial amount of cells, and can successfully extract the genome DNA by more than 500 cells; the method can be used for extracting the cell genome DNA in 96-well plates and 384-micro-well plates, and can finish the extraction of thousands of sample genome DNA in a short time and high flux; in addition, the method can also be used for extracting cell genome DNA in conventional cell culture dishes and 6, 12, 24 and 48 pore plates; the whole extraction process is carried out in the same cell culture container, a sample does not need to be transferred, and the extraction process does not need to be vibrated, so that the pollution among pores is avoided; the method is suitable for extracting genomic DNA of anchorage-dependent and suspension-grown cells; the method is safe and nontoxic and has extremely low cost; the genomic DNA extracted by the method is naked DNA and has no pollution of protein and RNA.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a simple, convenient and rapid high-throughput genome naked DNA extraction method.
Background
DNA is a main carrier of genetic information and is a main subject of research in the field of life sciences. The occurrence and progression of various diseases in humans are associated with variations in the genomic DNA of the host. The study of genomic variations and molecular mechanisms of disease occurrence is also a major research content in the life science field. Therefore, genomic DNA extraction is one of the most basic and important molecular biological experiments, and is a prerequisite for carrying out various detection means such as downstream PCR technology, southern blot, FISH and the like.
With the recent emergence of novel gene editing techniques, single base editing at the genomic DNA level has been achieved, and powerful tools have been provided for gene therapy of diseases related to genomic DNA mutations and the like. The current novel gene editing technology, including CRISPR-CAS9, greatly improves the efficiency of gene editing compared with the conventional technology, but still needs to screen hundreds, even thousands of colonies formed by single cells from which completely edited homozygotes are screened, wherein the most time-consuming step is to wait for the single cells to be amplified to a sufficient amount, and then extract the genomic DNA for downstream identification.
The currently available minigenome DNA extraction kit requires that the initial amount of cells is at least 2 ten thousand cells, and then the amplification from a single cell to 2 ten thousand cells requires at least 7 days even for tumor cells with a fast growth rate, and may require about 20 days for cells with a slow growth rate. Waiting for the cells to expand to a sufficient amount for extraction of genomic DNA is therefore a rate-limiting step of an experimental cycle.
In addition, most of the currently available genomic DNA extraction methods are based on magnetic beads, adsorption columns or phenol extraction and the like, phenol, isopropanol and the like are toxic organic solvents, and therefore, the method is harmful to workers and the environment and has a guidance of green science. Meanwhile, the methods need to transfer samples into different containers, the steps are complicated, the workload is still enough for dozens of samples, once hundreds of samples need to be processed simultaneously, the workload is too heavy, and the samples with huge number need to be transferred and marked, so that the samples are very easy to be mixed and polluted, a large amount of experimental consumables are consumed, a large amount of experimental wastes are generated, and the environmental pollution is caused.
In recent years, commercial reagents and methods capable of simultaneously extracting 96 samples have appeared, but all have the defects and shortcomings of high initial sample amount, sample transfer and the like. The initial amount of cells is generally required to be more than 5 thousands, samples need to be transferred into different containers, the steps are complicated, and the samples are easily polluted. In addition, the cost is extremely high, and more than thousand yuan of a genome DNA extraction kit for 96 samples causes great financial burden on scientific research work. In addition, no method for extracting genome DNA of cells in 384 micro-well plates exists, because the available genome DNA extraction methods all require high initial cell amount.
In order to simplify the experimental process, improve the experimental efficiency and accuracy and reduce the scientific research cost, the invention provides a simple and rapid extraction method of high-throughput genome DNA, which has low sample initial quantity and can process a large number of samples.
Disclosure of Invention
The present invention aims at solving the above problems and providing a simple and rapid method for extracting naked genomic DNA at high throughput.
The invention realizes the purpose through the following technical scheme:
the invention comprises the following steps:
s1: the growth and fusion rate of the anchorage-dependent growth cells in the orifice plate reaches 30-90%, and the cell culture solution is removed;
s2: adding cell lysis solution, placing in a wet box, and placing in an oven;
s3: taking out the pore plate from the wet box, adding DNA precipitation liquid after the temperature is reduced to room temperature, and standing;
s4: centrifuging by using a centrifuge, pouring the supernatant, adding a DNA cleaning solution, and centrifuging again; repeating the steps once;
s5: pouring the supernatant, placing in an ultra-clean bench, adding a DNA solution after the ethanol is completely volatilized, placing at room temperature or overnight in a refrigerator for dissolving, and determining the concentration and the purity of the genome DNA by an ultraviolet spectrophotometer and agarose gel electrophoresis;
s6: select a suitable volume of genomic DNA for subsequent experiments.
The invention has the beneficial effects that:
the method is a simple, convenient and quick high-throughput genome naked DNA extraction method, can be used for extracting the genome DNA of the mammalian cells, and compared with the prior art, the method is simple, convenient and quick, and greatly improves the extraction efficiency of the genome DNA; the sensitivity is high, the requirement on the initial amount of cells is extremely low, and more than 500 cells can successfully extract genome DNA; can be used for extracting cell genome DNA in 96-well plates and 384-micro-well plates, so that the extraction of genome DNA of thousands of samples can be completed in a short time and high flux; meanwhile, the method can also be used for extracting genomic DNA in cell culture plates such as conventional cell culture dishes, 6-hole plates, 12-hole plates, 24-hole plates, 48-hole plates and the like; the whole extraction process is carried out in the same cell culture container, a sample does not need to be transferred, and the extraction process does not need to be vibrated, so that the pollution among pores is avoided; the method is suitable for extracting genomic DNA of anchorage-dependent and suspension-grown cells; the method of the invention is safe, nontoxic and extremely low in cost, and the cost of the reagent for extracting the genome DNA of 96 samples is only about 10 yuan. In addition, the genomic DNA extracted by the method is naked DNA, has no pollution of protein, RNA and the like, and can be used for detection of methods such as downstream PCR amplification and southern blot.
Drawings
FIG. 1 is a photograph taken by a bright field microscope before and after addition of cells to a lysis solution;
FIG. 2 is an image of a brightfield microscope after addition of a DNA precipitation solution;
FIG. 3 is an agarose gel electrophoresis of genomic DNA;
FIG. 4 is a graph showing the effect of editing genomic DNA of cells in a well plate by the PCR method;
FIG. 5 is a diagram of genomic DNA extraction in a 6-well plate.
Detailed Description
The invention will be further described with reference to the accompanying drawings in which:
(1) the invention needs to prepare the following solutions: cell lysate (5-10 mM Tris/Cl pH 7.5, 5-10 mM EDTA, 5-10 mM NaCl, 0.2-1% SDS, 0.2-1 mg/ml proteinase K, 5-20 mug/ml RNase A); the DNA precipitation solution is absolute ethyl alcohol containing 5-10 mM NaCl. The DNA cleaning solution is 70% ethanol; DNA solution (10 mM Tris-HCl pH 8.0, 0.1mM EDTA). Note: the formulations of the solutions are working solution concentrations, protease K and RNase A in the cell lysate need to be added in fresh before the solution is used, and DNA precipitation solution and DNA cleaning solution need to be pre-cooled in a refrigerator at a temperature of-20 ℃ before the solution is used. And (5) storing the DNA dissolving solution in a refrigerator at 4 ℃.
(2) The cell source is 96 single cell clones obtained by gene editing through a CRISPR-CAS9 technology and screening through a flow cell sorting instrument. The cell type is human breast cancer cell line HCC1806 cells, and the cells are anchorage-dependent growth cells. And removing the cell culture solution when the cell growth fusion rate in the 96-well plate reaches about 30-90%. If the adherence of the cells is good, washing the cells once by using 100 mul PBS, and if the adherence is poor, discarding the supernatant of the culture solution.
(3) Adding 50 mul of cell lysate, placing the cell lysate into a wet box, and placing the cell lysate in a 55-degree oven for 15min-4h (figure 1). FIG. 1 is an image of a bright field microscope before and after a cell is added to a lysis solution, wherein a is an image of a cell under a 10-fold objective lens by using a bright field microscope before the cell is added to the lysis solution, b is an image of a cell under a 10-fold objective lens by using a bright field microscope after the cell lysis solution is added for 5min, and the result shows that the cell is very fully lysed within 5 min;
(4) the well plate is taken out of the wet box, 100 mul of DNA precipitation solution is added after the temperature is reduced to the room temperature, and the mixture is kept stand for 15min (figure 2). FIG. 2 is an image of a brightfield microscope after adding a DNA precipitation solution, and in FIG. 2, the DNA precipitation solution is added to the A5, B5 and C5 wells of a 96-well plate, and after being left at room temperature for 15min, the filamentous genomic DNA can be observed by imaging with a brightfield microscope under 4-fold and 20-fold objective lenses;
(5) centrifugation was carried out using a centrifuge at 3000rpm for 5 min.
(6) And pouring the supernatant, adding 150 mul of DNA cleaning solution, and centrifuging again. And repeating the steps once.
(7) After the supernatant is poured out, the supernatant is turned over on a water absorption paper towel and is lightly tapped to remove the ethanol to the maximum extent. And placing the mixture in an ultra-clean bench, adding 30 mul of DNA solution after the ethanol is completely volatilized, and placing the mixture at room temperature for 10min or overnight dissolving the mixture in a 4-DEG refrigerator. The concentration and purity of the genomic DNA were determined by UV spectrophotometer and agarose gel electrophoresis (FIG. 3). FIG. 3 is an agarose gel electrophoresis of genomic DNA. In FIG. 3, after the genomic DNA of FIG. 2 was dissolved, the DNA concentration and purity were measured by an ultraviolet spectrophotometer. The DNA concentrations in the A5, B5 and C5 wells are 278 ng/mul, 226 ng/mul and 293 ng/mul respectively, the OD260/OD280 are 1.85, 1.89 and 1.87 respectively, and the DNA concentrations and purities are high. Agarose gel electrophoresis was performed with a loading of approximately 50ng per well and imaging was performed by gel imaging equipment, which showed very good genomic integrity.
(8) Select a suitable volume of genomic DNA for subsequent experiments. If the PCR amplification template is used for PCR identification, 2 mu l can be taken as a PCR reaction template, and the amplification result is shown in figure 4. The effect of genomic DNA editing of cells in the well plate was identified by PCR method. A2 XHieff PCR Master Mix (YEASON) conventional PCR premixed solution is used, the amplification fragment of the used primer is about 350bp, the PCR reaction system is 10 mu l, and 5 mu l is used for agarose gel electrophoresis. N represents a negative control, i.e., a PCR reaction tube without the addition of a genomic DNA template; p represents a positive control, a PCR reaction tube to which a commercial genomic DNA template was added; A1-A11, B1-B11, C1-C11, D1-D11, E1-E11, F1-F11, G1-G11, H1-H11 and A12-H12 are PCR reaction tubes which are respectively added into a genome DNA template in a 96-well plate. The result shows that the negative control has no PCR band amplification, the positive control has 350bp PCR band amplification, 96 reaction tubes have band amplification, wherein the size of the amplified band is compared with that of the positive control, cells which are displaced are all cell clones after gene editing, and the sizes of the amplified bands are not obviously different compared with that of the positive control, and the amplified bands are identified by T7 nuclease endonuclease or first-generation sequencing.
The method is flexible, and can be placed in a refrigerator at 20 ℃ below zero overnight after the suspension of the steps 2, 4, 6 and 7.
The method of the invention has high extraction efficiency of genome DNA, if the genome DNA is extracted from cells cultured in a culture vessel with a bottom area larger than 4 square centimeters, such as a 6-well plate, and the like, the genome DNA can be visible with naked eyes after being added with DNA precipitation solution, and can be picked out by a sterilized toothpick and put into a 1.5ml centrifuge tube for subsequent cleaning and dissolving (figure 5). FIG. 5 is a drawing of genomic DNA extraction in 6-well plates, filamentous genomic DNA visible to the naked eye, which can be picked up and placed in 1.5ml centrifuge tubes for subsequent washing and lysis steps.
The method is suitable for adherent growth cells and suspension growth cells. The method and the steps aim at the anchorage-dependent growth cells in a 96-well plate, if the cells are suspension growth cells, the preparation concentration of the cell lysate is 6 times of the concentration of the working solution, 1/5 times of the cell lysate is added according to the initial volume of the cell culture solution, and the volume of the liquid in the subsequent steps is used according to the equal proportion of the steps. In addition to 96-well plates, the method of the present invention can also be used for extraction of genomic DNA in cell culture plates such as conventional cell culture dishes, 6-well plates, 12-well plates, 24-well plates, 48-well plates, and 394-well plates, and the solution in the method of the present invention can be used in an equal ratio according to the ratio of the cell culture area thereof to that of the 96-well plate.
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A simple, convenient and rapid high-throughput genome naked DNA extraction method is characterized by comprising the following steps:
s1: the growth and fusion rate of the anchorage-dependent growth cells in the orifice plate reaches 30-90%, and the cell culture solution is removed;
s2: adding cell lysis solution, placing in a wet box, and placing in an oven;
s3: taking out the pore plate from the wet box, adding DNA precipitation liquid after the temperature is reduced to room temperature, and standing;
s4: centrifuging by using a centrifuge, pouring the supernatant, adding a DNA cleaning solution, and centrifuging again; repeating the steps once;
s5: pouring the supernatant, placing in an ultra-clean bench, adding a DNA solution after the ethanol is completely volatilized, placing at room temperature or overnight in a refrigerator for dissolving, and determining the concentration and the purity of the genome DNA by an ultraviolet spectrophotometer and agarose gel electrophoresis;
s6: select a suitable volume of genomic DNA for subsequent experiments.
2. The simple, rapid and high-throughput naked genomic DNA extraction method according to claim 1, wherein: the cells of the step S1 are anchorage-dependent growth cells cultured in a 96-well plate, and if the anchorage-dependent growth cells are good, the cells are washed once by 100 mu l PBS, and if the anchorage-dependent growth cells are poor, cell lysis solution is directly added after culture solution supernatant is discarded.
3. The simple, rapid and high-throughput naked genomic DNA extraction method according to claim 1, wherein: the cell lysis solution of the step S2 comprises 5-10 mM Tris/Cl pH 7.5, 5-10 mM EDTA, 5-10 mM NaCl, 0.2-1% SDS, 0.2-1 mg/ml proteinase K and 5-20 mug/ml RNase A; the using amount of the cell lysate is 50 mu l, the temperature of an oven is 55 ℃, and the oven is placed for 15min-4 h.
4. The simple, rapid and high-throughput naked genomic DNA extraction method according to claim 1, wherein: the DNA precipitation solution in the step S3 is absolute ethyl alcohol containing 5-10 mM NaCl; the adding amount of the DNA precipitation solution is 100 mu l, and the standing time is 15 min.
5. The simple, rapid and high-throughput naked genomic DNA extraction method according to claim 1, wherein: centrifuging at 3000rpm in the step S4 for 5 min; the DNA cleaning solution is 70% ethanol; the addition amount of the DNA cleaning solution was 150. mu.l.
6. The simple, rapid and high-throughput naked genomic DNA extraction method according to claim 1, wherein: in the step S5, removing the ethanol by pouring the supernatant, then reversing the supernatant on a water absorption paper towel, and tapping to remove the ethanol to the maximum extent; and (4) placing in an ultra-clean bench, adding 30 mul of DNA solution after the ethanol is completely volatilized, and placing for 10min at room temperature or overnight dissolving in a 4-degree refrigerator.
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