CN111057704A - Dewaxing cracking combination liquid, kit and method for extracting DNA from paraffin section sample - Google Patents
Dewaxing cracking combination liquid, kit and method for extracting DNA from paraffin section sample Download PDFInfo
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Abstract
The invention provides a dewaxing and cracking combined liquid, a kit and a method for extracting DNA from a paraffin section sample, wherein the dewaxing and cracking combined liquid comprises the following components in concentration: 25-45 v/v% of liquid dewaxing reagent, 5-10 v/v% of absolute ethyl alcohol, 10-500 mM of Tris-HCl, 1-7M of guanidine hydrochloride, 2-5M, NaCl 0.1-1M, SDS 0.1.1-5 w/v% of urea, 1000.1-1 v/v% of triton X, 0.1-1 w/v% of spermidine and the balance of water. The dewaxing and cracking combined liquid can complete sample dewaxing, tissue cracking and DNA combining in one step, simplifies the operation steps, effectively saves the operation time and the labor cost, and has wide application range.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a dewaxing cracking combination solution, a kit and a method for extracting DNA (deoxyribonucleic acid) from a paraffin section sample.
Background
In the process of extracting DNA from paraffin section samples, because tissues are completely dehydrated and embedded in paraffin solid, nucleic acid in the tissues is tightly crosslinked with protein and paraffin, so that the extraction of the nucleic acid DNA from the paraffin section samples is very difficult. In general, nucleic acid is extracted from paraffin section samples, and then deparaffinization and rehydration of tissue samples, digestion of tissues by proteinase K, dissociation of lysates from DNA and proteins, and extraction and purification of DNA are required.
At present, most of methods and kits for extracting DNA from paraffin section samples are mainly operated manually, and the whole operation flow takes more than 4 hours; and although the single extraction flux is high, the whole workstation is expensive, the installation and operation cost is high, the applicability is not high, the automatic extraction device is not suitable for units with flexible scenes, such as scientific research institutions, laboratories of colleges and universities, related enterprises and public institutions and the like with low expenditure. In recent years, miniaturized, less-throughput semi-automatic nucleic acid extractors have been rapidly developed, and the miniaturized nucleic acid extractors have been widely used in the market.
The miniaturized automatic extraction instrument with less flux is characterized in that magnetic beads adsorbing DNA are transferred in buffer solutions only by moving a magnetic rod, the magnetic beads adsorbing DNA are uniformly mixed with reagent solutions by means of spiral vibration of the magnetic rod and the like, and finally the goal of nucleic acid extraction and purification is achieved. At present, the miniaturized semi-automatic nucleic acid extractor is widely applied to blood DNA extraction, saliva DNA extraction, oral swab DNA extraction and muscle tissue DNA extraction.
A conventional manual paraffin section sample DNA extraction kit or a paraffin section sample DNA extraction kit based on a liquid transfer working platform generally comprises the steps of ① dewaxing a paraffin section sample by xylene, manually sucking or mechanically sucking a certain volume of xylene solution into a reaction hole with the paraffin section sample, sucking and discarding the xylene solution after paraffin is dissolved, ② sucking absolute ethyl alcohol into the dewaxed paraffin section sample, washing the residual xylene solution, and then discarding absolute ethyl alcohol, a ③ heating device dries the residual absolute ethyl alcohol of FFPE, ④ manually or mechanically sucking protease K and tissue lysate by an instrument mechanical arm, adding the protease K and the tissue lysate into a sample tube at step ③, incubating at 50-60 ℃ for 30-60 minutes, then manually or mechanically sucking a certain volume of DNA binding solution by an instrument mechanical arm, adding the DNA binding solution into a solution at step ④, uniformly mixing the liquid in a suction head blowing manner, adding a certain volume of absolute ethyl alcohol by an ⑤, transferring all the liquid to a DNA membrane or adding magnetic beads for DNA adsorption, purifying, carrying out a washing step ⑥ to obtain a final liquid sample, and only needing no heavy operation of a large-scale, no-time, and no restriction of a large-time, and no-time, compared with the operation of a conventional laboratory operation, no-and no-required operation, no-required for the whole-required operation of a small-scale, no-time, no-required.
Disclosure of Invention
Based on the above, the invention provides a dewaxing cracking binding solution, a kit and a method for extracting DNA from paraffin section samples. The dewaxing and cracking combined liquid can complete sample dewaxing, tissue cracking and DNA combining in one step, simplifies the operation steps, effectively saves the operation time and the labor cost, and has wide application range.
The invention aims to provide a dewaxing cracking binding solution for extracting DNA from paraffin section samples.
The specific technical scheme is as follows:
a dewaxing cleavage binding solution for DNA extraction from paraffin section samples, said dewaxing cleavage binding solution comprising the following components in concentrations: 25-45 v/v% of liquid dewaxing reagent, 5-10 v/v% of absolute ethyl alcohol, 10-500 mM of Tris-HCl, 1-7M of guanidine hydrochloride, 2-5M, NaCl 0.1-1M, SDS 0.1.1-5 w/v% of urea, 1000.1-1 v/v% of triton X, 0.1-1 w/v% of spermidine and the balance of water.
In some of these embodiments, the liquid dewaxing agent is selected from one or more of mineral oil, paraffin oil, turpentine, silicone oil.
In some of these embodiments, the liquid dewaxing agent is a paraffinic oil.
In some embodiments, the concentration of guanidine hydrochloride in the dewaxed cleavage pool is between 3M and 6M.
In some of these embodiments, the dewaxed cleavage conjugate fluid comprises the following concentrations of components: 30-40 v/v% of liquid dewaxing reagent, 5-8.5 v/v% of absolute ethyl alcohol, 50-400 mM of Tris-HCl, 3-6M of guanidine hydrochloride, 2.5-4M, NaCl 0.2-0.8M, SDS 0.5.5-3.5 w/v% of urea, 0.2-0.8 w/v% of triton X-1000.2, 0.2-0.8 w/v% of spermidine and the balance of water.
In some embodiments, the pH of the dewaxing cleavage combined liquid is 4.5-6.5.
In some of these embodiments, it is preferred that the dewaxed cleavage conjugate further comprises proteinase K.
In some of these embodiments, the concentration of proteinase K in the dewaxed cleavage conjugate test solution is: 0.5-1.5 mg/mL.
In some of these embodiments, it is preferred that the concentration of proteinase K in the dewaxed cleavage conjugate test solution is: 0.8-1.2 mg/mL.
The invention also provides a preparation method of the dewaxing cracking binding solution for extracting the DNA of the paraffin section sample, which comprises the following steps:
(1) dissolving Tris-HCl, guanidine hydrochloride, urea, NaCl, SDS, triton X-100 and spermidine in water, and uniformly mixing to obtain a water phase reagent;
(2) adding the liquid dewaxing reagent into absolute ethyl alcohol, and uniformly mixing to obtain an oil phase reagent;
(3) and (3) adding the water phase reagent obtained in the step (1) into the oil phase reagent in the step (2), then adding the rest water, and uniformly mixing to obtain the dewaxing cracking binding solution.
In some embodiments, the preparation method of the dewaxing cleavage binding solution for extracting the DNA of the paraffin section sample comprises the following steps:
(1) dissolving Tris-HCl, guanidine hydrochloride, urea, NaCl, SDS, triton X-100 and spermidine in water, and uniformly mixing to obtain a water phase reagent;
(2) adding the liquid dewaxing reagent into absolute ethyl alcohol, and uniformly mixing to obtain an oil phase reagent;
(3) adding the water phase reagent obtained in the step (1) into the oil phase reagent obtained in the step (2), then adding the balance of water, and uniformly mixing;
(4) and (3) when the method is used for extracting the DNA of the paraffin section sample, adding proteinase K into the mixture obtained in the step (3), and uniformly mixing to obtain a dewaxing cleavage binding solution.
The invention also aims to provide a kit for extracting DNA from paraffin section samples.
The specific technical scheme is as follows:
a kit for extracting DNA from a paraffin section sample comprises a dewaxing cracking binding solution for extracting the DNA from the paraffin section sample and magnetic beads for adsorbing the DNA.
In some embodiments, the paraffin section sample DNA extraction kit further comprises a washing solution, a rinsing solution and a DNA eluent.
In some of these embodiments, the wash solution comprises the following concentrations of components: 10-70 w/v% of guanidine hydrochloride and 1001-20 v/v% of triton X-2.
In some of these embodiments, the rinse liquid comprises the following concentrations of components: 50-80 v/v% of absolute ethyl alcohol and 0.1-0.5M of NaCl.
In some of these embodiments, the DNA eluate is selected from the group consisting of TE buffer, EB buffer, ultrapure water.
The invention also aims to provide a method for extracting DNA from the paraffin section sample.
The specific technical scheme is as follows:
a method for extracting DNA from a paraffin section sample comprises the following steps:
(1) adding the dewaxing cleavage binding solution extracted from the DNA of the paraffin section sample into the paraffin section sample, uniformly mixing, and incubating at 60-70 ℃ for 60-80 min;
(2) adding magnetic beads for adsorbing DNA into the mixed solution obtained in the step (1), and uniformly mixing;
(3) separating the magnetic beads adsorbing the DNA in the step (2);
(4) adding a washing solution into the magnetic beads adsorbing the DNA for washing, then separating the magnetic beads adsorbing the DNA, and discarding the liquid;
(5) adding a rinsing liquid into the magnetic beads adsorbing the DNA for washing, then separating the magnetic beads adsorbing the DNA, and discarding the liquid;
(6) repeating the step (5) once;
(7) adding DNA eluent into the magnetic beads adsorbing the DNA for elution; separating the magnetic beads adsorbing the DNA, and collecting the liquid to obtain the DNA solution.
In some embodiments, the incubating step (1) at 60-70 ℃ for 60-80 min comprises: placing the mixture in a water bath kettle at the temperature of between 60 and 70 ℃ for 60 to 80 minutes, quickly taking out the mixture every 13 to 17 minutes, uniformly mixing the mixture in a vortex manner, and putting the mixture back into the water bath kettle again.
In some of these examples, step (1) was incubated at 65 ℃ for 70min, during which time it was quickly vortexed and replaced in a water bath again at 15min intervals.
In some embodiments, adding the magnetic beads for adsorbing DNA to the mixture obtained in step (1), and mixing, including: and (2) restoring the temperature of the mixture obtained in the step (1) to 15-30 ℃, adding magnetic beads for adsorbing DNA, uniformly mixing by vortex, and standing at room temperature for 13-18 min.
Compared with the prior art, the invention has the following beneficial effects:
1. the optimized paraffin section sample DNA extraction dewaxing and cracking combined liquid can complete sample dewaxing, tissue cracking and DNA combining in one step. And after dewaxing the paraffin section sample by using the dewaxing cracking binding solution, dividing the liquid into an oil phase and a water phase, dissolving paraffin in a dewaxing reagent to form an upper oil phase, cracking the sample, carrying out crosslinking decomposition on DNA, protein and paraffin, releasing the DNA, the protein and the paraffin into the lower water phase, adding magnetic beads for adsorbing DNA into the water phase, and washing and eluting the magnetic beads to obtain a DNA solution. When the dewaxing and cracking combined liquid is used for extracting the DNA of the paraffin section sample, the paraffin section sample dewaxing and rehydration, protease K digestion tissue, DNA and protein crosslinking by using a cracking liquid, DNA extraction and purification and other steps do not need to be performed step by step, so that the reagent is prevented from being added and the liquid is prevented from being removed for multiple times, the extraction steps are simplified, and the operation time and the labor cost are effectively saved.
2. The paraffin section sample DNA extraction dewaxing cracking combination solution provided by the invention is not only suitable for manual extraction, but also suitable for automatic DNA extraction instruments, including large-scale automatic workstations and small semi/full automatic nucleic acid extractors without liquid transfer function, and has a wide application range.
Drawings
FIG. 1 is a graph showing the results of 1% agarose gel electrophoresis of DNA solutions of paraffin section sample No. 1 extracted by the methods described in examples 6-7 and comparative example 1, wherein lane 1 is 15K DNA marker; lane 2 is DNA extracted as described in example 6; lane 3 is DNA extracted as described in example 7; lane 4 is DNA extracted by the method described in comparative example 1.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
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. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In one embodiment of the invention, a dewaxing cracking combination liquid for extracting DNA from paraffin section samples is provided, and the dewaxing cracking combination liquid comprises the following components in concentration: 25-45 v/v% of liquid dewaxing reagent, 5-10 v/v% of absolute ethyl alcohol, 10-500 mM of Tris-HCl, 1-7M of guanidine hydrochloride, 2-5M, NaCl 0.1-1M, SDS 0.1.1-5 w/v% of urea, 1000.1-1 v/v% of triton X, 0.1-1 w/v% of spermidine and the balance of water.
In some embodiments, the liquid dewaxing agent is an oil phase agent, and is a saturated hydrocarbon such as mineral oil, paraffin oil, turpentine oil and the like which can dissolve paraffin, and can also be any liquid dewaxing agent which is immiscible with the aqueous phase solution and can dissolve paraffin.
In some embodiments, the dewaxed cleavage conjugate solution for DNA extraction of paraffin section samples further comprises proteinase K; the concentration of the proteinase K in the dewaxing cleavage binding solution for extracting the paraffin section sample DNA is as follows: 0.5 to 1.5mg/mL, more preferably 0.8 to 1.2 mg/mL. The proteinase k can promote other components in the dewaxing cleavage binding solution to better exert the effect and improve the DNA yield. However, proteinase K is an active enzyme substance, which cannot be stored in the binding solution without dewaxing and cracking by a strong denaturing agent for a long time, and therefore can be added when needed.
In some embodiments, the concentration of guanidine hydrochloride in the dewaxing cleavage combined liquor is 3M to 6M.
In some embodiments, 1.0-2.0 mL of the dewaxing cleavage binding solution is added to 1-10 paraffin section samples, and 0.2-0.25 mL of the dewaxing cleavage binding solution is added to more than 10 paraffin section samples, for paraffin section samples with a length of 3cm, a width of 1cm and a thickness of 5 um.
The dewaxing cracking combination liquid is in a uniform liquid state before use, and after paraffin section samples are dewaxed, the liquid can be separated into a water phase and an oil phase due to the existence of paraffin. Paraffin is dissolved in the dewaxing reagent to form an upper oil phase, the sample is cracked, and DNA is uncrosslinked with protein and paraffin, so that the DNA is released into a lower water phase. The aqueous phase component comprises Tris-HCl, guanidine hydrochloride, urea, NaCl, SDS, triton X-100 and spermidine. Wherein Tris-HCl is a pH buffering agent, and the buffering range is 4.5-6.5; guanidine hydrochloride is a strong protein denaturant; urea is a protein dissolving agent; NaCl provides a certain ionic strength for the aqueous phase solution; SDS is an anionic surfactant, can promote protein dissolution, the peptide chain breaks; triton X-100 is a nonionic surfactant and can stabilize the activity of proteinase K; spermidine can stabilize DNA, prevent further DNA breakage in high temperature environment, and promote the combination of DNA and magnetic beads.
Another embodiment of the present invention provides a method for extracting DNA from a paraffin section sample, comprising the following steps:
(1) adding the dewaxing cleavage binding solution extracted from the DNA of the paraffin section sample into the paraffin section sample, uniformly mixing, and incubating at 60-70 ℃ for 60-80 min;
(2) adding magnetic beads for adsorbing DNA into the mixed solution obtained in the step (1), and uniformly mixing;
(3) separating the magnetic beads adsorbing the DNA in the step (2);
(4) adding a washing solution into the magnetic beads adsorbing the DNA for washing, then separating the magnetic beads adsorbing the DNA, and discarding the liquid;
(5) adding a rinsing liquid into the magnetic beads adsorbing the DNA for washing, then separating the magnetic beads adsorbing the DNA, and discarding the liquid;
(6) repeating the step (5) once;
(7) adding DNA eluent into the magnetic beads adsorbing the DNA for elution; separating the magnetic beads adsorbing the DNA, and collecting the liquid to obtain the DNA solution.
In some embodiments, the incubating step (1) at 60 ℃ to 70 ℃ for 60min to 80min comprises: placing the mixture in a water bath kettle at the temperature of between 60 and 70 ℃ for 60 to 80 minutes, quickly taking out the mixture every 13 to 17 minutes, uniformly mixing the mixture in a vortex manner, and putting the mixture back into the water bath kettle again; more preferably, it comprises: incubate at 65 ℃ for 70min, during which time it is quickly vortexed and replaced in a water bath again every 15 min.
In some embodiments, the adding of the magnetic beads for adsorbing DNA to the mixture of step (1) and mixing uniformly includes: and (2) restoring the temperature of the mixture obtained in the step (1) to 15-30 ℃, adding magnetic beads for adsorbing DNA, uniformly mixing by vortex, and standing for 13-18 min. The magnetic beads have a weak ability to adsorb DNA at a temperature higher than 30 ℃.
EXAMPLE 1A Paraffin cleavage binding solution for DNA extraction from Paraffin section samples
The present embodiment relates to a dewaxing cleavage binding solution for DNA extraction of paraffin section sample, which comprises the following components in concentration: 35 v/v% of paraffin oil, 7 v/v% of absolute ethyl alcohol, 250mM of Tris-HCl, 3M guanidine hydrochloride, 0.5 4M, NaCl 0.5 w/v% of urea, 0.5M, SDS 3 w/v% of triton X-1000.5 v/v% of spermidine and the balance of water.
The preparation method of the dewaxing cracking binding solution for extracting the paraffin section sample DNA comprises the following steps:
(1) dissolving Tris-HCl, guanidine hydrochloride, urea, NaCl, SDS, triton X-100 and spermidine in water, and uniformly mixing to obtain a water phase reagent;
(2) adding paraffin oil into absolute ethyl alcohol, and uniformly mixing to obtain an oil phase reagent;
(3) and (3) adding the water phase reagent obtained in the step (1) into the oil phase reagent in the step (2), then adding the rest water, and uniformly mixing to obtain the dewaxing cracking binding solution.
Example 2 Paraffin cleavage binding solution for DNA extraction of Paraffin section sample
The present embodiment relates to a dewaxing cleavage binding solution for DNA extraction of paraffin section sample, which comprises the following components in concentration: 35 v/v% of paraffin oil, 7 v/v% of absolute ethyl alcohol, 250mM of Tris-HCl, 3M guanidine hydrochloride, 0.5 4M, NaCl 0.5 w/v% of urea, 0.5M, SDS 3 w/v% of triton X-1000.5 v/v%, 0.5 w/v% of spermidine, 1mg/mL of protease and the balance of water.
The preparation method of the dewaxing cracking binding solution for extracting the paraffin section sample DNA comprises the following steps:
(1) dissolving Tris-HCl, guanidine hydrochloride, urea, NaCl, SDS, triton X-100 and spermidine in water, and uniformly mixing to obtain a water phase reagent;
(2) adding paraffin oil into absolute ethyl alcohol, and uniformly mixing to obtain an oil phase reagent;
(3) adding the water phase reagent obtained in the step (1) into the oil phase reagent obtained in the step (2), then adding the balance of water, and uniformly mixing;
(4) and (3) when the method is used for extracting the DNA of the paraffin section sample, adding proteinase K into the mixture obtained in the step (3), and uniformly mixing to obtain a dewaxing cleavage binding solution.
Example 3A kit for extracting DNA from a paraffin section sample
The present embodiment provides a kit for extracting DNA from a paraffin section sample, comprising the dewaxing lysis binding solution described in embodiment 1, 50mg/mL magnetic beads for adsorbing DNA, a washing solution, a rinsing solution, and a DNA eluent;
the wash solution contained the following concentrations of components: 50 w/v% of guanidine hydrochloride and X-10015 v/v% of triton.
The rinse liquid comprises the following components in concentrations: 70 v/v% of absolute ethanol and 0.3M of NaCl.
The DNA eluent is EB buffer solution.
Example 4 kit for extracting DNA from paraffin section sample
The present embodiment provides a kit for extracting DNA from a paraffin section sample, comprising the dewaxing lysis binding solution described in embodiment 2, 50mg/mL magnetic beads for adsorbing DNA, a washing solution, a rinsing solution, and a DNA eluent;
the wash solution contained the following concentrations of components: 50 w/v% of guanidine hydrochloride and X-10015 v/v% of triton.
The rinse liquid comprises the following components in concentrations: 70 v/v% of absolute ethanol and 0.3M of NaCl.
The DNA eluent is EB buffer solution.
Example 5 method for extracting DNA from paraffin section sample
The method for extracting the DNA of the paraffin section sample is a manual extraction method, the kit in the embodiment 3 is used for extraction, and the specific steps are as follows:
(1) transferring the paraffin section sample to a 1.5mL centrifuge tube without nuclease, adding 1.0mL sample dewaxing cleavage binding solution, uniformly mixing by vortex, placing in a 65 ℃ water bath for 70 minutes, rapidly taking out the paraffin section sample at intervals of 15 minutes, uniformly mixing by vortex, and placing back in the water bath again;
(2) taking out the centrifuge tube, cooling to 25 deg.C, adding 30ul of magnetic bead solution (50mg/mL) for adsorbing DNA, mixing by vortex, and standing at room temperature for 15 min;
(3) placing the centrifugal tube on a magnetic frame and standing for 5min to enable the magnetic beads to be completely adsorbed on the tube wall;
(4) completely removing the liquid by using a pipettor, taking down the centrifugal tube from the magnetic frame, adding 1.0mL of washing liquid, vortexing for 1min, fully resuspending the magnetic beads, placing on the magnetic frame for 5min, and removing all the liquid after the liquid is clarified;
(5) taking the centrifugal tube in the previous step down from the magnetic frame, adding 1.0mL of rinsing liquid, carrying out vortex resuspension on magnetic beads, then placing the centrifugal tube on the magnetic frame for 1-2 min, and removing all liquid after the liquid is clarified;
(6) repeating the step (5) once, then keeping the centrifugal tube on the magnetic frame, opening the cover for 5min, and volatilizing residual liquid;
(7) taking down the centrifugal tube from the magnetic frame, adding 60ul of DNA eluent EB buffer solution, mixing the magnetic beads evenly by vortex, and placing for 5min at room temperature; and finally, placing the centrifugal tube on a magnetic frame, standing for 2min, and carefully transferring the liquid to a new nuclease-free centrifugal tube to obtain the DNA solution.
Example 6 method for extracting DNA from paraffin section sample
The method for extracting the DNA of the paraffin section sample is a manual extraction method, and the kit of embodiment 4 is used for extraction, and the specific steps are the same as those of embodiment 5.
Example 7 method for extracting DNA from paraffin section sample
The method for extracting the DNA of the paraffin section sample comprises the following steps of automatically extracting by using a magnetic rod type automatic nucleic acid extractor which is manufactured by Shanghai Baydid company and has the model of GenePure-32, wherein the kit for extraction is the kit in the embodiment 4:
(1) adding 1.0mL of dewaxing lysis binding solution to the A1 well position of a 96 deep-well plate, adding 30ul of magnetic bead solution (50mg/mL) adsorbing DNA to the A2 well position, adding 1.0mL of washing solution to the A3 well position, adding 1.0mL of rinsing solution to the A4 and A5 wells, and adding 65ul of nucleic acid eluent EB buffer solution to the A6 well position;
(2) the program is as follows, ① A1 hole heating temperature is 65 ℃, heating time is 70min, mechanical arm up-and-down vibration intensity is 4 grades, vibration amplitude is 0-90%, ② A2 hole is set with magnetic bar magnetic attraction time 30s, transferring to A1 hole, magnetic bar descending amplitude is to liquid bottom, magnetic attraction time is 0 (release magnetic bead), mechanical arm up-and-down vibration intensity is 6 grades, amplitude is 0-100%, duration time is 15min, ③ A1 hole is set with magnetic bar magnetic attraction time 1min, transferring to liquid bottom in A3 hole, release magnetic bead, mechanical arm up-and-down vibration intensity is 9 grades, vibration time is 10min, magnetic attraction time 1min is then set, transferring to A4 hole, ④ A4, A5 hole setting program and parameter are consistent with A3 hole, ⑤ A6 hole setting parameter is magnetic bar staying 3min, then descending to eluent bottom, magnetic bead releasing, mechanical arm up-and down vibration intensity is set with 10 grades, duration time is 5 min.
(3) After the program of the instrument is set, placing the paraffin section sample in an A1 hole position of a 96 deep-hole plate, then placing the 96 deep-hole plate in the specified position of the instrument, and starting the instrument to start automatic DNA extraction;
(4) after the operation of the instrument is finished, transferring 60ul of DNA solution in the A6 hole to a new nuclease-free centrifuge tube to obtain the DNA solution.
Comparative example 1 method for extracting DNA from paraffin section sample
The method for extracting the DNA of the paraffin section sample is an existing conventional manual extraction method, and extraction is carried out by utilizing a Qiagen NFPE tissue DNA extraction kit (product number 56404), and the method comprises the following specific steps:
(1) opening a thermostat and heating to 45 ℃;
(2) transferring the paraffin section sample to a 1.5mL centrifuge tube;
(3) adding 1mL of dimethylbenzene into the centrifuge tube, uniformly mixing the dimethylbenzene with the centrifuge tube by oscillation, centrifuging the mixture at the room temperature (15-25 ℃) of 12000rpm for 2 minutes, and removing supernatant; if paraffin is still observed, repeating the steps until no paraffin exists;
(4) adding 1mL of absolute ethyl alcohol into the centrifuge tube, oscillating and uniformly mixing, centrifuging at the room temperature of 12000rpm for 2 minutes, and removing the supernatant;
(5) opening the centrifugal tube, and placing the centrifugal tube in a thermostat at 45 ℃ for incubation for 15-30 minutes to completely volatilize ethanol;
(6) adding 180 mu L of lysate ATL into the 1.5mL centrifuge tube, uniformly mixing by oscillation, performing instantaneous centrifugation, adding 20 mu L of protease K, uniformly mixing by oscillation, and performing instantaneous centrifugation;
(7) placing the 1.5mL centrifuge tube in a thermostat, and incubating at 56 ℃ for 1-1.5 hours, or prolonging the time until the sample is completely cracked;
(8) quickly transferring the 1.5mL centrifuge tube into a thermostat preheated to 90 ℃ and incubating for 1 hour;
(9) the elution column was equilibrated at room temperature for 10 min;
(10) adding 200 mu L of binding solution AL into the 1.5mL centrifuge tube in the step (8), uniformly mixing by oscillation, and performing instantaneous centrifugation;
(11) adding 200 mu L of absolute ethyl alcohol into the 1.5mL centrifuge tube, uniformly mixing by oscillation, and carrying out instantaneous centrifugation;
(12) transferring all the liquid in the 1.5mL centrifuge tube into the elution column in the step (9), centrifuging at the room temperature of 12000rpm for 1 minute, discarding the collection tube and the waste liquid, taking out the elution column, and placing the elution column into a new collection tube;
(13) adding 500 μ L of prepared cleaning solution AW1 into the elution column, centrifuging at 12000rpm at room temperature for 1min, discarding the collection tube and waste liquid, taking out the elution column, and placing in a new collection tube;
(14) adding 500 μ L of prepared cleaning solution AW2 into the elution column, centrifuging at 12000rpm at room temperature for 1min, discarding the collection tube and waste liquid, taking out the elution column, and placing in a new collection tube;
(15) centrifuging the collecting tube with the elution column for 3 minutes at room temperature of 12000rpm, discarding the collecting tube and the waste liquid, taking out the elution column and placing the elution column into a new 1.5mL centrifuge tube;
(16) opening the tube cover of the elution column, standing for 2 minutes at room temperature to volatilize the absolute ethyl alcohol;
(17) and (3) suspending 60 mu L of eluent in the center of the elution column membrane, covering the cover of the elution column, standing for 2 minutes at room temperature, centrifuging for 1 minute at 12000rpm at room temperature, and eluting until the liquid in a 1.5mL centrifuge tube is the extracted DNA solution.
Taking 40 paraffin slice samples, taking 4 paraffin slices as one group, and numbering samples 1-10 respectively in 10 groups. Dividing each paraffin section sample in each group into 4 parts, then respectively merging one fourth of each paraffin section sample to obtain 4 parts of paraffin section samples, respectively extracting DNA of the 4 parts of paraffin section samples in each group by using the extraction methods in the examples 5-7 and the comparative example 1 to obtain DNA solutions, respectively taking 5ul of the DNA solutions to perform 1% agarose gel electrophoresis detection, and simultaneously taking 1ul of the DNA solutions to perform Nanodrop detection on the concentration and purity of the DNA, wherein the specific results are shown in Table 1 and figure 1 (taking sample No. 1 as an example):
TABLE 1 DNA concentration and purity test results
Note: the concentration in the table is given in ng/ul.
As can be seen from Table 1, the methods described in examples 5-7 can successfully extract DNA from paraffin sections. Compared with example 5, the method of examples 6 and 7 has higher DNA yield, because the dewaxing lysis binding solution in the kit used in examples 6 and 7 contains proteinase K, proteinase K can promote other components in the dewaxing lysis binding solution to better exert effect, and the DNA yield is improved. The DNA solutions extracted in examples 6 and 7 have the same concentration and fragment range (FIG. 1, sample No. 1 as an example) and better purity than those extracted in comparative example 1 (the conventional method for extracting DNA from paraffin section samples). However, the extraction steps of the extraction method of the invention are less than those of the extraction method of the invention, the extraction time required by the extraction method of the invention is about 4h, while the extraction time required by the extraction method of the invention of examples 5 to 7 is about 2h, which is only half of the extraction time of the extraction method of the invention of the comparison example 1, thereby effectively simplifying the operation steps, shortening the extraction time and improving the extraction efficiency. In addition, the kit of the present invention is not only suitable for manual extraction (examples 5 and 6), but also suitable for a small semi/full-automatic nucleic acid extractor without pipetting function (example 7), and has a wide range of applications.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
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 present 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 (17)
1. A dewaxing and cracking combined liquid for extracting DNA (deoxyribonucleic acid) of a paraffin section sample is characterized by comprising the following components in concentration: 25-45 v/v% of liquid dewaxing reagent, 5-10 v/v% of absolute ethyl alcohol, 10-500 mM of Tris-HCl, 1-7M of guanidine hydrochloride, 2-5M, NaCl 0.1-1M, SDS 0.1.1-5 w/v% of urea, 1000.1-1 v/v% of triton X, 0.1-1 w/v% of spermidine and the balance of water.
2. The liquid dewaxing cleavage binding solution for DNA extraction of paraffin section samples of claim 1, wherein the liquid dewaxing reagent is selected from one or more of mineral oil, paraffin oil, turpentine oil and silicone oil.
3. The liquid paraffin-splitting reagent of claim 2, wherein the liquid paraffin-splitting reagent is paraffin oil.
4. The dewaxing lysis solution for DNA extraction of paraffin section sample according to claim 1, wherein the concentration of guanidine hydrochloride in the dewaxing lysis solution is 3M to 6M.
5. The paraffin section sample DNA extraction dewaxing lysis binding solution according to any one of claims 1 to 4, wherein the dewaxing lysis binding solution comprises the following components in concentration: 30-40 v/v% of liquid dewaxing reagent, 5-8.5 v/v% of absolute ethyl alcohol, 50-400 mM of Tris-HCl, 3-6M of guanidine hydrochloride, 2.5-4M, NaCl 0.2-0.8M, SDS 0.5.5-3.5 w/v% of urea, 0.2-0.8 w/v% of triton X-1000.2, 0.2-0.8 w/v% of spermidine and the balance of water.
6. The liquid for dewaxing and splitting DNA extraction from a paraffin section sample according to any one of claims 1 to 5, wherein the pH of the liquid for dewaxing and splitting is 4.5 to 6.5.
7. The paraffin section sample DNA extraction dewaxing cleavage binding solution as set forth in any one of claims 1 to 5, wherein the dewaxing cleavage binding solution further comprises proteinase K.
8. The dewaxing lysis binding solution for DNA extraction of paraffin section sample according to claim 7, wherein the concentration of proteinase K in the dewaxing lysis binding solution is: 0.5-1.5 mg/mL.
9. The method for preparing a binding solution for dewaxing and cracking of a paraffin section sample for DNA extraction according to any one of claims 1 to 6, comprising the steps of:
(1) dissolving Tris-HCl, guanidine hydrochloride, urea, NaCl, SDS, triton X-100 and spermidine in water, and uniformly mixing to obtain a water phase reagent;
(2) adding the liquid dewaxing reagent into absolute ethyl alcohol, and uniformly mixing to obtain an oil phase reagent;
(3) and (3) adding the water phase reagent obtained in the step (1) into the oil phase reagent in the step (2), then adding the rest water, and uniformly mixing to obtain the dewaxing cracking binding solution.
10. A kit for extracting DNA from a paraffin section sample, comprising the binding solution for dewaxing cleavage for DNA extraction from a paraffin section sample according to any one of claims 1 to 8, and magnetic beads for adsorbing DNA.
11. The kit for extracting DNA from a paraffin section sample according to claim 10, further comprising a washing solution, a rinsing solution, and a DNA eluent.
12. The DNA extraction kit for paraffin section samples according to claim 11, wherein the washing solution contains the following components at the following concentrations: 10-70 w/v% of guanidine hydrochloride and 1001-20 v/v% of triton X-2.
13. The paraffin section sample DNA extraction kit of claim 11, wherein the rinsing solution comprises the following components in concentration: 50-80 v/v% of absolute ethyl alcohol and 0.1-0.5M of NaCl.
14. A method for extracting DNA from a paraffin section sample is characterized by comprising the following steps:
(1) adding the dewaxing cracking binding solution extracted from the DNA of the paraffin section sample of any one of claims 1 to 8 into the paraffin section sample, uniformly mixing, and incubating at 60-70 ℃ for 60-80 min;
(2) adding magnetic beads for adsorbing DNA into the mixed solution obtained in the step (1), and uniformly mixing;
(3) separating the magnetic beads adsorbing the DNA in the step (2);
(4) adding a washing solution into the magnetic beads adsorbing the DNA for washing, then separating the magnetic beads adsorbing the DNA, and discarding the liquid;
(5) adding a rinsing liquid into the magnetic beads adsorbing the DNA for washing, then separating the magnetic beads adsorbing the DNA, and discarding the liquid;
(6) repeating the step (5) once;
(7) adding DNA eluent into the magnetic beads adsorbing the DNA for elution; separating the magnetic beads adsorbing the DNA, and collecting the liquid to obtain the DNA solution.
15. The method for extracting DNA from a paraffin section sample according to claim 14, wherein incubating at 60-70 ℃ for 60-80 min comprises: placing the mixture in a water bath kettle at the temperature of between 60 and 70 ℃ for 60 to 80 minutes, quickly taking out the mixture every 13 to 17 minutes, uniformly mixing the mixture in a vortex manner, and putting the mixture back into the water bath kettle again.
16. The method for extracting DNA from a paraffin section sample as claimed in claim 15, wherein the incubation is carried out at 65 ℃ for 70min, and the sample is rapidly taken out, vortexed, mixed and placed back in the water bath again every 15 min.
17. The method for extracting DNA from a paraffin section sample according to any one of claims 14 to 16, wherein the adding of magnetic beads for absorbing DNA to the mixture of step (1) and the mixing comprises: and (2) restoring the temperature of the mixture obtained in the step (1) to 15-30 ℃, adding magnetic beads for adsorbing DNA, uniformly mixing by vortex, and standing for 13-18 min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500704A (en) * | 2020-04-28 | 2020-08-07 | 广州市金圻睿生物科技有限责任公司 | Human fetus chromosome aneuploidy detection kit and method |
| CN113308460A (en) * | 2020-12-15 | 2021-08-27 | 深圳未知君生物科技有限公司 | Kit for extracting bacterial DNA in paraffin section |
| CN113527408A (en) * | 2021-07-08 | 2021-10-22 | 谱天（天津）生物科技有限公司 | Method for high-throughput extraction of proteomes of different types of samples |
| CN113755486A (en) * | 2021-09-23 | 2021-12-07 | 翌圣生物科技(上海)股份有限公司 | Paraffin remover for processing paraffin-embedded samples and method for extracting nucleic acid from paraffin-embedded samples |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575220A (en) * | 2009-09-03 | 2012-07-11 | 贝克顿·迪金森公司 | Methods and compositions for direct chemical lysis |
| CN105247065A (en) * | 2013-03-15 | 2016-01-13 | 雅培分子公司 | Compositions and methods for nucleic acid extraction |
| CN107475251A (en) * | 2017-09-19 | 2017-12-15 | 成都诺博森生物技术有限公司 | A kind of lysate and its application in tissue or cell, extraction RNA is preserved |
| CN108124451A (en) * | 2015-07-24 | 2018-06-05 | 塞弗德公司 | For extracting the composition and method of DNA and RNA from tissue sample |
| CN109022418A (en) * | 2018-08-17 | 2018-12-18 | 上海迈景纳米科技有限公司 | A kind of fast dewaxing method that FFPE sample nucleic acid extracts |
| CN109321523A (en) * | 2018-10-16 | 2019-02-12 | 厦门艾德生物医药科技股份有限公司 | A kind of blood additive |
| CN110050070A (en) * | 2016-12-22 | 2019-07-23 | 文塔纳医疗系统公司 | Full-automatic method for extracting nucleic acid for tissue sample |
-
2019
- 2019-12-31 CN CN201911415475.6A patent/CN111057704B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575220A (en) * | 2009-09-03 | 2012-07-11 | 贝克顿·迪金森公司 | Methods and compositions for direct chemical lysis |
| CN105247065A (en) * | 2013-03-15 | 2016-01-13 | 雅培分子公司 | Compositions and methods for nucleic acid extraction |
| CN108124451A (en) * | 2015-07-24 | 2018-06-05 | 塞弗德公司 | For extracting the composition and method of DNA and RNA from tissue sample |
| CN110050070A (en) * | 2016-12-22 | 2019-07-23 | 文塔纳医疗系统公司 | Full-automatic method for extracting nucleic acid for tissue sample |
| CN107475251A (en) * | 2017-09-19 | 2017-12-15 | 成都诺博森生物技术有限公司 | A kind of lysate and its application in tissue or cell, extraction RNA is preserved |
| CN109022418A (en) * | 2018-08-17 | 2018-12-18 | 上海迈景纳米科技有限公司 | A kind of fast dewaxing method that FFPE sample nucleic acid extracts |
| CN109321523A (en) * | 2018-10-16 | 2019-02-12 | 厦门艾德生物医药科技股份有限公司 | A kind of blood additive |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500704A (en) * | 2020-04-28 | 2020-08-07 | 广州市金圻睿生物科技有限责任公司 | Human fetus chromosome aneuploidy detection kit and method |
| CN111500704B (en) * | 2020-04-28 | 2023-10-27 | 广州市金圻睿生物科技有限责任公司 | Kit and method for detecting human fetal chromosome aneuploidy |
| CN113308460A (en) * | 2020-12-15 | 2021-08-27 | 深圳未知君生物科技有限公司 | Kit for extracting bacterial DNA in paraffin section |
| CN113527408A (en) * | 2021-07-08 | 2021-10-22 | 谱天（天津）生物科技有限公司 | Method for high-throughput extraction of proteomes of different types of samples |
| CN113527408B (en) * | 2021-07-08 | 2023-08-25 | 谱天（天津）生物科技有限公司 | Method for high-throughput extraction of different types of sample proteomes |
| CN113755486A (en) * | 2021-09-23 | 2021-12-07 | 翌圣生物科技(上海)股份有限公司 | Paraffin remover for processing paraffin-embedded samples and method for extracting nucleic acid from paraffin-embedded samples |
| CN114891778A (en) * | 2022-03-31 | 2022-08-12 | 杭州丹威生物科技有限公司 | Method and kit for full-automatic extraction of DNA of paraffin-embedded tissue section |
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