CN1187196A - Method and apparatus for DNA extraction - Google Patents
Method and apparatus for DNA extraction Download PDFInfo
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- CN1187196A CN1187196A CN96194606A CN96194606A CN1187196A CN 1187196 A CN1187196 A CN 1187196A CN 96194606 A CN96194606 A CN 96194606A CN 96194606 A CN96194606 A CN 96194606A CN 1187196 A CN1187196 A CN 1187196A
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Abstract
Described is a method and apparatus for DNA extraction from the cell suspension. The said method uses a hollow membrane filter to separate the DNA and cell debris after the cell cracking. The cell suspension can be a suspension of cultured cells or cells contained in body fluids such as blood. An ion exchange process is included in this method so as to purify the DNA. The container has a hollow membrane filter (5) combined at its terminals. The said method can be used to extract genomic DNA from cells, which is particularly applicable to extract plasmid DNA from microbes.
Description
The present invention relates to be used for extracting the method and apparatus of DNA from cell suspension.Specifically, the present invention relates to extract plasmid DNA and extract the method and apparatus of genomic dna from microorganism and zooblast from microorganism.
Background technology
The application of recombinant DNA technology is usually included in the clone of target DNA or the operation and uses plasmid or clay.Usually, carry the microorganism of plasmid or clay and extract plasmid or clay increase plasmid or clay from culture at last by cultivation.The DNA that extracts usually needs purifying.
For some recombinant DNA technology, particularly DNA purifying and order-checking have been developed automatically or semi-automatic program, and many methods still need manual operations.The situation of the method for extracting for plasmid DNA particularly.Ordinary method for example alkaline bleach liquor cleavage or boiling lysis (referring to, people such as Sambrook, molecular cloning: laboratory manual, second edition, cold spring harbor laboratory publishes, cold spring port, New York, 1989) comprises many steps.Whether it successfully depends on researchist's state of the art.Therefore, can cause the result who produces variant by unskilled experimenter to the repeated application of described method.
Often situation about occurring is to extract plasmid or cosmid DNA from many different cultures.Owing in some recombinant DNA technologies, use the droplet plate, so require in this case from become hundred kinds of different cultures, to extract DNA.Conventional extraction procedure is not suitable for automatic operation, or even semi-automatic operation.Therefore, extraction plasmid or cosmid DNA are the operations big with labour intensity consuming time from each a large amount of cultures.
Therefore need a kind of method that can be used for extracting plasmid or cosmid DNA from microorganisms cultures, this method is easier and have more repeatability than existing program.Also need a kind of extracting method of operation automatically that is adapted to, so that can process a large amount of cultures.
Equally, recombinant DNA technology and DNA analysis The Application of Technology relate to the genomic dna that uses animal blood and other body fluid.Ordinary method for example use organic solvent extracting method (referring to, people such as Sambrook, molecular cloning: laboratory manual, second edition, cold spring harbor laboratory publishes, the cold spring port, New York, 1989) comprise many steps, whether it successfully depends on researchist's state of the art.Therefore, can cause the result who produces variant by unskilled experimenter to the repeated application of described method.
Often situation about occurring is to extract genomic dna from a large amount of samples, particularly for the situation of using DNA in polymerase chain reaction and dna sequencing.Owing in some recombinant DNA technologies and genetic analysis, use the droplet plate, so require in this case from become hundred kinds of samples, to extract DNA.
Therefore need a kind of method that can be used for extracting genomic dna from blood and other body fluid, this method is easier and have more repeatability than existing program.Also need a kind of extracting method of operation automatically that is adapted to, so that can process a large amount of samples.
Summary of the invention
One object of the present invention is to provide the method for extracting DNA from cell suspension, and this method has overcome the shortcoming that exists in the existing extracting method.Specifically, one object of the present invention is to provide the method for optionally extracting plasmid DNA from cell suspension, and this method has overcome the existing shortcoming that is used to extract the method for plasmid DNA.
Another object of the present invention provides the device that is used for extracting from cell suspension DNA.
Above and hereinafter the term " cell suspension " that uses comprises the culture of zooblast, microorganisms cultures, and body fluid is blood for example, lymph liquid, urine and seminal fluid and individual cells other dispersion system in body fluid matrix.
Above and hereinafter the term " plasmid " that uses is meant the dna molecular that is referred to as plasmid and clay.
According to the first embodiment of the present invention, provide the method for extracting DNA from cell suspension, this method comprises the following steps:
1) cell suspension is added in the filtration unit with hollow membrane filter;
2) if desired, cross the substratum that filters to remove the described cell that suspends;
3) add cracked solution to described cell, be incubated the sufficiently long time of described cell, so that released dna; With
4) filter the cracked solution that contains described DNA.
Second embodiment according to the present invention provides the method for extracting DNA from cell suspension, and this method comprises the following steps:
1) cell suspension is added in the filtration unit with hollow membrane filter;
2) if desired, cross the substratum that filters to remove the described cell that suspends;
3) add cracked solution to described cell, be incubated the sufficiently long time of described cell, so that released dna; With
4) filter the cracked solution that contains described DNA;
5) filtrate with step (4) joins in the Ion Exchange Medium;
6) with the described Ion Exchange Medium of first solution washing with material wash-out that will be except that DNA; With
7) with the described Ion Exchange Medium of second solution washing with the described DNA of wash-out.
Third embodiment according to the present invention provides the method for extracting plasmid DNA from microorganisms cultures, and this method comprises the following steps:
1) microorganisms cultures that will carry plasmid DNA is added in the filtration unit with hollow membrane filter;
2) if desired, filter to remove substratum excessively;
3) cracked solution is added to described microorganism and be incubated the sufficiently long time of described microorganism therefrom to discharge plasmid DNA; With
4) filter the cracked solution that contains described plasmid DNA.
According to the 4th embodiment of the present invention, the method for extracting plasmid DNA from microorganisms cultures is provided, this method comprises the following steps:
1) microorganisms cultures that will carry plasmid DNA is added in the filtration unit with hollow membrane filter;
2) if desired, filter to remove substratum excessively;
3) add cracked solution to described microorganism, be incubated the sufficiently long time of described microorganism, so that discharge plasmid DNA; With
4) filter the cracked solution that contains described plasmid DNA;
5) filtrate with step (4) joins in the Ion Exchange Medium;
6) with the described Ion Exchange Medium of first solution washing with material wash-out that will be except that plasmid DNA; With
7) with the described Ion Exchange Medium of second solution washing with the described plasmid DNA of wash-out.
According to the 5th embodiment of the present invention, the device that is used for culturing cell and extracts DNA is provided, this device comprises:
Container with an opening end and a closed end, this closed end comprise the hollow membrane filter of the liquid communication that allows described internal tank and outside; With
Seal the movable sealing thing of described closed end.
According to the 6th embodiment of the present invention, the device that is used for culturing cell and extracts DNA is provided, this device comprises a plurality of the 5th described devices of embodiment.
According to the 7th embodiment of the present invention, the test kit that is used for culturing cell and extracts DNA is provided, this test kit comprises at least a the 5th described device of embodiment or at least a the 6th the described device of embodiment.
Term in this area " hollow membrane filter " and the commutative use of term " hollow fiber filter ".But, only use last term herein.
With reference to first embodiment of the present invention, the principle that can recognize this method is the relevant cell of cracking and utilizes the hollow membrane filter to isolate the lysate that contains DNA from cell debris.Utilize this method to extract genomic dna from the culture of zooblast and microorganism and these cells.As hereinafter describing in detail, also can utilize this method to extract plasmid DNA from microorganism.But, when being used to extract plasmid DNA, use relatively mild cracking condition.This method has very large advantage than known DNA extraction program, and wherein whole procedure is finished in the hollow membrane filtration unit.
With regard to the first step of the described method of first embodiment, any cell of cracked that is adapted to the step (3) of this method all can be used for whole procedure.If at first make it to disperse to form suspension, then also the cell of cohesion can be used for this program.The method that the cell that condenses is formed dispersion liquid is that those skilled in that art are known.As mentioned above, this cell suspension can be the suspension of culturing cell.Utilize ordinary method and condition can prepare described culture.The culture of Shi Yonging can be the other culture samples that produces or As described in detail below in the methods of the invention, can produce in the device that comprises the hollow membrane filter.Therefore, be the in-situ preparing culture in back one situation.
Excessive suspension substratum can be filtered out, on the hollow membrane filter, keep spissated cell (step of this method (2)).Add malleation or add negative pressure by inlet side and can realize filtering in outlet one side of membrane filter at membrane filter.By centrifugal or be the method that typically adds malleation by using air pressure.Usually negative pressure can provide by vacuumizing, and this is preferred filter method.Filter method in the past also is applicable to the later step of first embodiment, and be applicable to second embodiment the institute in steps.
The method of first embodiment further comprises the step (2a) that the cell collected on the vacuum diaphragm filter with step (2) washs.The washing soln that is applicable to step (2a) also is that those skilled in that art are known, generally includes the buffered soln of sucrose or glucose.Preferred washing soln comprises:
16.5% sucrose
36mM?Tris.HCl(pH8.0)
55mM?EDTA
Washing soln also comprises the enzyme of fracturing cell walls composition.Described enzyme is N,O-Diacetylmuramidase normally.
By under malleation or condition of negative pressure, the washing soln of certain volume can be finished the washing of pair cell through membrane filter.The volume of used washing soln is not important, most convenient be the original volume that is about as much as cell suspension.
Preferably a kind of damping fluid that contains chaotropic agent that is with or without stain remover of the cracked solution of the step of first embodiment (3).Suitable chaotropic agent comprises Guanidinium hydrochloride, sodium iodide, the salt of sodium perchlorate and guanidine for example, guanidine thiocyanate.Usually chaotropic agent exists with the concentration of 3-6M.Even can use the saturated solution (about 8M under some situation) of chaotropic agent.Suitable stain remover comprises Tween
TM20, TritonX-100
TM, Nonidet
TMP-40, Brij58
TM, Sodium desoxycholate, N-Sarkosyl L or the like.Usually stain remover exists with the concentration of 0.05-5%.TritonX-100 for example
TMCan be present in the cracked solution with the concentration of 0.75-5%.The pH of cracked solution is 5-9 normally.Also can contain for example urea of denaturing agent in the cracked solution.
Preferred cracked solution comprises:
The 4M guanidine thiocyanate
0.1M sodium acetate (pH5.0)
5％TritonX-100
TM
This cracked solution of 3M urea is specially adapted to from animal cell extraction genomic dna.
Preferably use the cracked solution of minimum volume can avoid diluting the DNA of extraction.Usually, use the cracked solution volume that is equivalent to culture volume approximately.
The insulation sufficiently long time of cell is to discharge a large amount of genomic dnas in the presence of cracked solution.For zooblast, soaking time 3-15 minute is suitable.
Filter the cracked solution of the DNA that contains extraction at the final step of first embodiment of the present invention.Can utilize the known technology concentration of DNA of those skilled in that art or DNA is transferred to another kind of solution.For example, utilize the organic solution can deposit D NA or the solution of plasmid DNA carried out gel-filtration or dialysis.
The step (3) of the method for first embodiment and (4) are repeated one or repeatedly to improve the output of DNA.The multiple step only relates to and adds fresh cracked solution, and the insulation regular hour to be discharging other DNA, and filters cracked solution.
The method of second embodiment provides the ordinary method that is used to obtain purify DNA.Four steps of the beginning of this method can comprise optional step (2a) by the above described enforcement of first embodiment.The Ion Exchange Medium that is applicable to this method steps (5) is that those skilled in that art are known, and comprises with known groups DEAE (diethylaminoethyl-) for example the inert base that QAE (QAE) and Q (quaternary ammonium) replace.Preferred Ion Exchange Medium is a silicon-dioxide.
Be designed to allow DNA to be attached on the Ion Exchange Medium cracked solution of step (3), wash away other compound that is contained in the lysate.For combining with the silicon-dioxide filter, particularly preferred cracked solution is the cracked solution that defines in the step (3) of above-mentioned first embodiment.
First solution that the washing Ion Exchange Medium generally includes several parts sees through this medium.First solution is a kind of solution of dissolving DNA not preferably.For being used in combination with the silicon-dioxide Ion Exchange Medium, 80% aqueous isopropanol soluble in water is preferred.
See through this medium by a kind of solution and can realize that the step 8 couple DNA of this method carries out wash-out solubilized DNA.Typical solutions comprises the water that contains low concentration of salt, or TE (pH7.4-8.0) (TE solution is to produce the aqueous solution of being made up of 10mM Tris.HCl and 1mM EDTA from pH7.0-9.0Tris.HCl stoste and the preparation of pH8.0EDTA stoste).
The 3rd and the 4th described method of embodiment of the present invention is substantially the same respectively in the method for first and second embodiments, and different is that two kinds of initial methods are applicable to the microorganism use.For the first step of the method for third and fourth embodiment, any cracked microorganism that is adapted to the step (3) of this method all can be used for whole procedure.But microorganisms cultures is a bacterial cultures usually.Utilize ordinary method and condition to prepare microorganisms cultures.As the embodiment of front, the culture of Shi Yonging can be the other culture samples that produces or As described in detail below in the method, can produce in the device that comprises the hollow membrane filter.Therefore, be the in-situ preparing culture in back one situation.
The cracked solution of the step of the method for third and fourth embodiment (3) can comprise a kind of damping fluid that contains chaotropic agent that is with or without stain remover equally.The chaotropic agent of above-mentioned qualification and stain remover also can be used for extracting plasmid DNA from microorganism.Preferred chaotropic agent comprises that concentration is respectively Guanidinium hydrochloride and the guanidine thiocyanate of 4-6M and 3-5M.Preferred cracked solution comprises:
The 6M Guanidinium hydrochloride
0.75％TritonX-100
TM
200mM HEPES (pH6.5) or 100mM Tris.HCl (pH6.4).
The insulation sufficiently long time of microorganism still should make the amount of the chromosomal DNA that discharges and other cellular material be reduced to minimum to discharge a large amount of plasmid DNA in the presence of cracked solution.Soaking time also depends on the type of microorganism.For bacterium intestinal bacteria for example, soaking time 3-15 minute is suitable.
As the embodiment of front, the step (3) and the step (4) of the method for third and fourth embodiment can be repeated to improve the output of plasmid DNA.Will be appreciated that further other detailed method of first and second embodiments also can be used for the method for third and fourth embodiment.
The 5th embodiment, the cylinder that the container of device is normally elongated are described now.This container can be any volume, but for the preferably about 2 milliliters volume of the device that is used for auto-programming.Usually the hollow membrane filter approximately be no more than hold syringe long-pending 25% and can comprise many finger pieces or the reticulation that extends in the container, or in container axially extended single cylindrical element.
The sealer that is used for the closed end of tightness system can be a kind of sheet metal sealer that pulls out, or cooperates or the lid of frictional fit form seal end with screw.
In preferred embodiments, this device is also included within the movable sealing thing of the opening end of container.Usually the sealing thing is gas-pervious lid, so that microorganism aerobic is cultivated.
The configuration that will be appreciated that this device should add substratum.To substratum, this device of insulation is so that culturing cell under appropriate condition with required microbial inoculant.Handle culture according to the extraction procedure of first embodiment then.
In addition, this device can be used to cultivate virulent phage (for example, M13 or λ).Inoculate phage with host microorganism.After cultivating, extract the substratum that contains phage particle through the filtration of hollow membrane filter and can separate phage from host microorganism.
Will be appreciated that also device of the present invention can be used for directly extracting DNA from cell, also promptly cultivate dispensable in position for the extraction procedure cell.
The 5th described device of embodiment is preferably from there being the material preparation of resistance to organic solvent and conventional sterilising technology.Suitable material comprises polypropylene, polystyrene and acrylate plastics.
The 6th the described device of embodiment generally includes the linear combination of the 5th the described device of embodiment, and wherein each container is elongated cylinder.Preferably, this device has 8 containers to constitute so that corresponding with the hole of 8 * 12 hole droplet plates of standard, and this makes this device can be used for automatic operation.
A plurality of containers in the device combination of the 6th embodiment can be linked together by means of brittle or solid reticulation.Another kind of optionally method can be adjacent with the part of the wall of an a kind of part of wall of a container and adjacent container.
Preferably the device of the 5th and the 6th embodiment is transformed the filtration unit that contains Ion Exchange Medium with joint at the opening end of container.This can make the cracked solution that contains plasmid DNA be directly delivered to Ion Exchange Medium, thereby promotes the purifying of DNA.In case the DNA that extracts is attached to Ion Exchange Medium, this device breaks away from filtration unit, and implements remaining DNA purification step according to step (6) and (7) of second and the 4th embodiment.
The particularly preferred mode of utilizing the device of the 6th embodiment is and the collecting tubule combination that is suitable for being connected vacuum pump.Preferably collecting tubule includes the dish of the lid that contains a plurality of holes.Usually this lid has the linear combination of 8 holes with the device that receives the 6th embodiment at least.Preferably, this is stamped 96 holes of droplet panel configuration.
When being used in combination, device is inserted in the hole of collecting tubule lid with collecting tubule.Add stopper so that use negative pressure untapped covering.Then if necessary in step (1)-(3) of the method for first to the 4th embodiment, vacuumize by collecting tubule and to finish filtration step.
Collect the DNA that extracts if desired, on the collecting tubule dish droplet plate is being installed so that in the set pore, collect from the cracked solution that contains DNA of device combination sucking-off.
Also collecting tubule can be connected with the device of the 6th embodiment and be used for the DNA that purifying extracts.In step (1)-(3) of implementing second and the 4th embodiment afterwards, between device combination and collecting tubule lid, can insert the ion-exchange filtration unit with implementation step (4).Remove device combination implementation step (5) and (6) then, at last the DNA of droplet plate with wash-out during collecting step (7) is being installed on the collecting tubule dish.
Will be appreciated that above-described process can control the release that is used for implementing extracting with the reagent of each step of purge process automatically, be controlled at application of negative pressure on the collecting tubule automatically.
Except that being used to extract the device of DNA, the test kit of the 7th embodiment can comprise to be implemented to extract and the reagent of each step of purge process, and/or is used for the filtration unit of purification step.
Test kit also can comprise the device combination that links to each other with the vacuum collecting tubule.Foregoing test kit further can comprise the filtration unit combination.
The accompanying drawing summary
Fig. 1 is the cross-section side view that comprises the device of single culture vessel and filter.This figure also illustrates the filter that is used for the DNA purifying.
Fig. 2 is the cross-section side view that comprises the device of a plurality of culture vessels and filter, has shown the DNA purifying filter that connects.
Fig. 3 is the orthographic plan of observing from the top of vacuum collecting tubule.
Fig. 4 is the sectional drawing of vacuum collecting tubule.
Fig. 5-10 has described the gel of ethidium bromide staining, is used to analyze the DNA sample that comes from various extraction steps.
Implement best way of the present invention and alternate manner
At first describe device of the present invention with reference to the accompanying drawings. For Fig. 1, shown to comprise be used to connecing Receive the cylinder part 2 of culture medium 3. This device contains the closed end 4 that comprises hollow membrane filter 5. The sealer of lid 6 forms can frictional fit mode be installed to closed end 4, in order to stop Liquid in the device passes the hollow membrane filter and loses.
The total diameter of the device that Fig. 1 describes is 40 millimeters * 8 millimeters OD, is to use polypropylene Or the polystyrene preparation.
The funnel type filter 9 that wherein has silica net 10 is shown in device 1 time The side. Can see that from this figure the diameter that reduces end 4 is so that the openend of terminal 4 accesss to plant 9 11. The wall of cylinder part 2 and the frictional fit of installing between 9 the openend 11 provide anti-Leak sealing.
Describe now Fig. 2, wherein shown the linear combination by 8 installation compositions of Fig. 1 21. Device remains in the combination by net, and wherein a kind of net is instructed in 22. Right In the DNA purifying, combination 22 is used in combination with the filter combination 23 of Fig. 1.
Vacuum concetrated pipe 31 when Fig. 3 has described from top the observation. Can see that concetrated pipe has 8 Round, every row has 12 holes.
The cross-section side view of the concetrated pipe that is shown in Fig. 3 is provided in Fig. 4. Can see Lid 32 and dish 33. Being provided with parts 34 is used for being connected with the vavuum pump of being indicated by arrow 35. Figure 3 and 4 concetrated pipe 31 is to be different from the scale of installation drawing 2.
The configuration of the concetrated pipe that Fig. 3 and 4 describes is convenient to the extraction for DNA. At this In the configuration, between combination 21 and concetrated pipe 31, inserted the filter combination 23 of Fig. 2. The funnel shaped part of each filter has neck 24, is fixed in the hole of concetrated pipe lid 32, and Form sealing.
Be understood that when with it fixedly the time combination 23 can occupy cover owning of a row of 32 The hole. Device combination 21 can be fixed to combination 23 for the DNA purifying then. By Be fixed to the combination 21 and 23 of concetrated pipe lid 32, make up from device by the silica filter Each container sucking liquid and enter into the dish 33.
Also can recognize and the droplet plate can be placed the chamber 36 of concetrated pipe 31 in order to will pass through the hole Liquid collection in the particular bore of droplet plate.
Be non-limiting embodiment below.Embodiment 1
The extraction of plasmid DNA and purifying
In this serial experiment, utilize the method for second embodiment to estimate the effect of various cracked solution.
The intestinal bacteria DH10B that carries plasmid pGem5Zf-is inoculated in LB (LuriaBertani) substratum and according to standard program spends the night 37 ℃ of shaking culture.As funnel 1 ml sample of incubated overnight is added to 0.2 micron Dynagard by means of 5 milliliters of injection needle tubes
TMME hollow membrane filtration unit.The outlet that the glass fibre filtration unit is fixed to the hollow membrane filtration unit is to form the filter set.Described filtration unit is that those skilled in that art are known, can buy by commercial sources.
Vacuumizing by the outlet in the filter set can be with cell fixation to the hollow membrane filter.For cracking fixed cell and release plasmid DNA, 1 milliliter cracked solution is added to filtered set, make it with immobilized cell room temperature insulation 5 minutes.The cracked solution that is detected contains chaotropic agent (Guanidinium hydrochloride, guanidine thiocyanate or sodium iodide) and stain remover (TritonX-100
TMOr Brij58
TM, Sodium desoxycholate) combination.In this step, plasmid DNA discharges and enters the solution that makes DNA be attached to glass fibre filter from cell.Then solution is gathered by filter, thereby promptly by the tubular fibre membrane filter with then by glass fibre filter-plasmid DNA is fixed to glass fibre filter.
In low salt buffer, before the wash-out plasmid DNA, remove the hollow fiber filter part of filter set, keep glass fibre filter.Then under vacuum condition with 1 milliliter 80% Virahol, 10mM HEPES (pH6.5) washs this filter three times.Low-salt aqueous solution or TE with 100 microlitres under vacuum condition join the DNA of vacuum drying glass fibre filter with the wash-out purifying.
The plasmid DNA of said procedure purifying is carried out agarose gel electrophoresis and detected the DNA bands of a spectrum by ethidium bromide staining.In order to compare, also the pGem5Zf-to the preparation of intestinal bacteria alkaline bleach liquor cleavage analyzes.The alkaline bleach liquor cleavage method of employed plasmid DNA preparation is described in people such as Sambrook (seeing above).
Stained gel is depicted in Fig. 5,25% the total plasmid amount by 1 milliliter microbial culture deposits yields that swimming lane 2-9 has gone up sample.Sample below analyzing: swimming lane 1, the Spp-1 molecular weight marker thing of EcoRI digestion; Swimming lane 2 uses the contrast of the plasmid purification pGem5Zf-of alkaline bleach liquor cleavage acquisition; Swimming lane 3 is with 3.2M guanidine thiocyanate and 0.5%TritonX-100
TMThe pGem5Zf-of cracking preparation; Swimming lane 4 is with 3.2M guanidine thiocyanate and 0.5%Brij58
TMPGem5Zf-with 0.2% Sodium desoxycholate cracking preparation; Swimming lane 5 is with 4.8M Guanidinium hydrochloride and 0.5%TritonX-100
TMThe pGem5Zf-of cracking preparation; Swimming lane 6 is with 4.8M Guanidinium hydrochloride and 0.5%Brij58
TMPGem5Zf-with 0.2% Sodium desoxycholate cracking preparation; Swimming lane 7 is used the 4.5M sodium iodide, 90mM S-WAT and 0.5%TritonX-100
TMThe pGem5Zf-of cracking preparation; Swimming lane 8 is used the 4.5M sodium iodide, 90mM S-WAT, 0.5%Brij58
TMPGem5Zf-with 0.2% Sodium desoxycholate cracking preparation.
Can be clear that from Fig. 5 although high yield as the alkaline bleach liquor cleavage program is not provided, the cracked solution that detects can reclaim highly purified plasmid DNA in second method embodiment.Contain 4.8M Guanidinium hydrochloride and 0.5%TritonX-100
TMCracked solution as if effective especially.The detection of embodiment 2 cracking conditions
Utilization contains the TritonX-100 of 4.8M Guanidinium hydrochloride and various concentration
TMCracked solution further test.Various cracking conditions are also detected.Other experiment condition and step are referring to the description of embodiment 1.Adopt agarose gel electrophoresis that 25% sample from total yield plasmid of 1 milliliter of bacterial cultures is analyzed.
Fig. 6 shows the result of agarose gel analysis.Analyze following sample: swimming lane 1, with the Spp-1 molecular weight marker thing of EcoRI digestion; Swimming lane 2 and 3 uses the contrast of the plasmid purification pGem5Zf-of alkaline bleach liquor cleavage acquisition; Swimming lane 4 is with containing 1%TritonX-100
TMThe pGem5Zf-of cracked solution cracking preparation in 5 minutes; Swimming lane 5 is with containing 2%TritonX-100
TMThe pGem5Zf-of cracked solution cracking preparation in 5 minutes; Swimming lane 6 is with containing 1%TritonX-100
TMThe pGem5Zf-of two cracking in 5 minutes preparations of cracked solution insulation; Swimming lane 7 is with containing 2%TritonX-100
TMThe pGem5Zf-of two cracking in 5 minutes preparations of cracked solution insulation; Swimming lane 8, under vacuum condition with constant flow velocity with containing 1%TritonX-100
TMThe pGem5Zf-of cracked solution cracking preparation in 5 minutes; With swimming lane 9, under vacuum condition with constant flow velocity with containing 2%TritonX-100
TMThe pGem5Zf of cracked solution cracking preparation in 5 minutes.
Fig. 6 result displayed as can be seen, the output and the purity that are used for the cracked solution of swimming lane 6 and 7 and the plasmid DNA that cracking condition provides are equivalent to the alkaline bleach liquor cleavage program basically.Embodiment 3
Washing is to the influence of yield plasmid
Carrying out a series of experiment estimates the effect of washing bacterial cell before cleavage step.Washing soln contains 16.5% sucrose, 36mM Tris.HCl (pH8.0) and 55mMEDTA, at room temperature insulation different time of bacterial cell in washing soln.By containing the Guanidinium hydrochloride of 4.8M, 1%TritonX-100
TMIn the solution of 160mM HEPES (pH6.5), the insulation cell was implemented the cracking of bacterial cell in 5 minutes under room temperature.Other experiment condition and step are referring to the description of the foregoing description 1.As the embodiment of front, adopt agarose gel electrophoresis that 25% sample from total yield plasmid of 1 milliliter of bacterial cultures is analyzed.
Fig. 7 has described the result of agarose gel electrophoresis, the sample below wherein analyzing: swimming lane 1, the Spp-1 molecular weight marker thing of EcoRI digestion; Swimming lane 2 uses the contrast of the plasmid purification pGem5Zf-of alkaline bleach liquor cleavage acquisition; Swimming lane 3 is with washing soln insulation 5 minutes, cracked pGem5Zf-then; Swimming lane 4 is with washing soln insulation 30 minutes, cracked pGem5Zf-then; Swimming lane 5 is with washing soln insulation 5 minutes, cracked pGem5Zf-then; Swimming lane 6 is with washing soln insulation 30 minutes, cracked pGem5Zf-then.
During the preparation of the plasmid DNA that swimming lane 3 and 4 is analyzed, by filtration unit with-2kPa sucking-off solution, and during the preparation of swimming lane 5 and 6 plasmid DNA of analyzing, use-13kPa.
Fig. 7 shows that the described method of above-mentioned embodiment makes the output of plasmid DNA preparation and purity be equivalent to for example alkaline bleach liquor cleavage program of conventional procedure at least.The great advantages of described implementation process is to utilize single covering device can implement this method, and does not need to shift the solution that contains DNA.Embodiment 4 extracts and purified genomic dna from blood
In a series of experiments, utilize the genomic dna that from bovine blood, reclaims to estimate the effect of various cracked solution.
As antithrombotics, join the TE of 400 microlitres with the bovine blood that contains the moisture EDTA of 0.1% (weight/volume) of 100 microlitre volumes.This provides the erythrocytic instrument that exists in the cracking blood (protoheme cracking).This solution is joined 5cm
20.2 micron polypropylene hollow membrane filter, described filter with 100% ethanol pre-wetted so that solution stream is crossed this filter.From other material for example the mixture of rhodia and nitrocellulose form such filter and can buy by commercial sources, and work with the same manner.The outlet that the glass fibre filtration unit is fixed to the hollow membrane filtration unit is to form the filter set.
Vacuumize and from protoheme cracked blood, to be fixed to the hollow membrane filter with containing the nuclear cell by outlet in the filter set.For cracking fixed cell and release genomic dna, 1 milliliter cracked solution is added to the filter set, make it with immobilized cell room temperature insulation 5 minutes.The cracked solution that is detected contains the 4M guanidine thiocyanate, 5%TritonX-100
TM, 0.1M sodium acetate (pH6.5) and concentration are from the urea of 0.5M-4M.In this step, discharge genomic dna and enter the solution that makes DNA be attached to glass fibre filter from the nuclear of cell.This step repeats 3 times, with complete released dna.
Then under vacuum condition with 1 milliliter 80% Virahol, 10mM Tris.HCl (pH6.4) washs this filter three times.Low salts solution with the TE (pH8.5) of 100 microlitres under vacuum condition joins the genomic dna of glass fibre filter with the wash-out purifying.
The genomic dna of purifying is carried out agarose gel electrophoresis and detects the DNA bands of a spectrum by ethidium bromide staining.For comparison, also analysis and utilization Progen Progenome
TMThe genomic dna of I genomic dna purification kit (from Progen Industrial Co., Ltd, 2806 IpswichRoad, Darra, Queensland 4076, Australia obtains) preparation is analyzed.
Fig. 8 has described stained gel.Swimming lane 2-7 has gone up sample 25% the total genomic dna that produces from the bovine blood of 100 microlitres.Analyze following sample: swimming lane 1, the Spp-1 molecular weight marker thing of EcoRI digestion; Swimming lane 2 uses Progenome
TMThe contrast of the genomic dna of I purifying; Swimming lane 3 is with the genomic dna of the cracked solution cracking preparation that contains the 0.5M urea; Swimming lane 4 is with the genomic dna of the cracked solution cracking preparation that contains the 1M urea; Swimming lane 5 is with the genomic dna of the cracked solution cracking preparation that contains the 2M urea; Swimming lane 6 is with the genomic dna of the cracked solution cracking preparation that contains the 3M urea; Swimming lane 7 is with the genomic dna of the cracked solution cracking preparation that contains the 4M urea.
Do not providing as Progenome
TMDuring the same high yield of I genomic dna purification kit, the cracked solution that is detected provides highly purified genomic dna preparation.Contain 4M Guanidinium hydrochloride and 5%TritonX-100
TM, as if the cracked solution of the urea of 0.1M sodium acetate pH5.0 and concentration 3M effective especially.The comparison of embodiment 5 cultural methods
The intestinal bacteria DH10B that carries plasmid pMW5 be inoculated into the LB substratum and according to standard program 37 ℃ of vibration incubated overnight.(pMW5 be make up by Progen Industrial Co., Ltd and comprise part lambda particles phage among the pUC19).In order to detect various cultural methods, also with this inoculation to the LB substratum and be with or without under the aeration condition at 0.2 micron (5cm
2) in the polypropylene hollow fiber filtration unit in 37 ℃ of overnight incubation.Can realize the ventilation of culture to the outlet of hollow fiber filter device by applying the adjustable compression air.Ventilation is the bubble that passes substratum of visible form.
After the insulation of spending the night, the type culture of 250 microlitres is applied to the hollow membrane filtration unit, this device is moistening so that permission solution stream strainer with 100% ethanol in advance.The hollow membrane filter is a device of using the same type of original position cultivation as mentioned above.So analysis is subsequently carried out with standard model, remove the original position overnight culture from the hollow membrane filter appts, 250 microlitres partly are added to are similar to the moistening in advance similar fresh device of 100% ethanol.But in fact, the original position culture filters at the hollow membrane filtration unit that is used for cultivating, and does not transfer to fresh device.The outlet that the glass fibre filtration unit is fixed to the hollow membrane filtration unit is to form the filter set.
Vacuumizing by the outlet in the filter set can be with cell fixation to the hollow membrane filter.Before the bacterium cracking and discharging plasmid DNA, with 1 milliliter 16.5% the sucrose of containing, the solution washing cell of 40mM Tris.HCl (pH8.0) and 60mMEDTA contains the 6M Guanidinium hydrochloride by application, 0.1M Tris.HCl (pH6.4), 0.75%TritonX-100
TMSolution carry out the cracking of bacterial cell and the release of plasmid DNA, at room temperature be incubated 5 minutes with fixed cell.The solution that will contain plasmid DNA under vacuum condition is gathered sucking-off from filter then, thereby plasmid DNA is fixed to glass fibre filter.Repeat cleavage step then so that at utmost reclaim plasmid DNA.
Before glass fibre filter wash-out plasmid DNA, remove the hollow fiber filter part of filtered set, keep glass fibre filter.Then under vacuum condition with 1 milliliter 80% Virahol, 10mM Tris.HCl (pH6.4) washing back one filter three times.Low salts solution (pH8.5) with the TE of 100 microlitre volumes under vacuum condition joins the DNA of vacuum drying glass fibre filter with the wash-out purifying.
Carry out agarose gel electrophoresis and detect the DNA bands of a spectrum by the plasmid DNA of said procedure purifying with ethidium bromide staining.
Stained gel is depicted in Fig. 9.Swimming lane 2-4 has gone up sample total plasmid of 25% from the microbial culture deposits yields of 250 microlitres.Sample below analyzing: swimming lane 1, with the Spp-1 molecular weight marker thing of EcoRI digestion; Under the swimming lane 2, not ventilation situation in 0.2 micron hollow fiber filter device in the plasmid DNA of the bacteria purification of 37 ℃ of overnight incubation; Swimming lane 3, under the ventilation situation in 0.2 micron hollow fiber filter device in the plasmid DNA of the bacteria purification of 37 ℃ of overnight incubation; Swimming lane 4 is according to the plasmid DNA of standard method at the bacteria purification of 37 ℃ of overnight incubation.
Can be clearly seen that from Fig. 9 the bacterium of cultivating does not hinder the recovery of plasmid in hollow fiber filter device under the ventilation situation.It is possible that this result confirms the identical hollow membrane filter culturing bacterium that is used for DNA extraction step subsequently.Embodiment 6 is from cultured animals cell extraction DNA
Carrying out a series of experiment estimates from the genomic dna that the mammalian cell of cultivating reclaims utilizing extraction step of the present invention.
People's colon-cancer cell (HCT-116 that will in McCoys 5a substratum, cultivate; ATCC preserving number CCL 247) suspension is added to 0.2 micron Dynagard by means of 5 milliliters of injection needle tubes as funnel
TMThe ME hollow fiber filter device.The outlet that the glass fibre filtration unit is fixed to the hollow membrane filtration unit is to form the filter set.
By using vacuum cell is fixed to hollow fiber filter from solution in the outlet of filter set.For lysing cell and release genomic dna, 1 milliliter cracked solution is added to the filter set, make it with immobilized cell room temperature insulation 5 minutes.
Cracked solution contains 4M guanidine thiocyanate, 5%TritonX-100
TM, 0.1M sodium acetate (pH5.0) and 3M urea.In this step, discharge genomic dna and enter the solution that makes DNA be attached to glass fibre filter from cell.This step repeats 3 times, with at utmost from the fixed cell released dna.
Before glass fibre filter wash-out genomic dna, remove the hollow fiber filter part, keep the set of glass fibre filter.Then under vacuum condition with 1 milliliter 80% Virahol, 10mM Tris.HCl (pH6.4) washs this filter three times.Low salts solution (pH8.5) with the TE of 100 microlitre volumes under vacuum condition joins the genomic dna of glass fibre filter with the wash-out purifying.
Genomic dna is carried out agarose gel electrophoresis and detects the DNA bands of a spectrum with ethidium bromide staining.
Stained gel is depicted in Figure 10.Swimming lane 2-5 25% the total genomic dna output that gone up sample wherein.Sample below analyzing: swimming lane 1, with the Spp-1 molecular weight marker thing of EcoRI digestion; Swimming lane 2 is from the genomic dna of 1 milliliter of suspension cracking people colon-cancer cell preparation; Swimming lane 3 is from the genomic dna of 2 milliliters of suspension cracking people colon-cancer cell preparations; Swimming lane 4 and 5 is from the genomic dna of 5 milliliters of suspension cracking people colon-cancer cell preparations.
The result who is depicted in Figure 10 shows that extracting method of the present invention can be applied to the cultured animals cell effectively.
Will be appreciated that and can do many modifications to aforesaid method and device, and can not break away from wide boundary of the present invention and scope, described boundary and scope are defined by the appended claims.
Claims (62)
1. extract the method for DNA from cell suspension, described method comprises the following steps:
1) cell suspension is added in the filtration unit with hollow membrane filter,
2) if desired, cross the substratum that filters to remove the described cell that suspends;
3) add cracked solution to described cell, be incubated the sufficiently long time of described cell, so that released dna; With
4) filter the cracked solution that contains described DNA.
2. method according to claim 1, wherein said DNA is a genomic dna.
3. method according to claim 1, wherein said cell suspension are the cultured animals cells.
4. method according to claim 1, wherein said cell suspension are to be selected to comprise blood, lymph liquid, the body fluid of seminal fluid and urine.
5. method according to claim 1 comprises further that wherein washing is collected in the step of the cell on the hollow fiber filter in step (2).
6. method according to claim 1, wherein said cracked solution comprises the buffered soln of chaotropic agent.
7. method according to claim 6, wherein said chaotropic agent is selected from Guanidinium hydrochloride, guanidine thiocyanate, sodium iodide and sodium perchlorate.
8. method according to claim 6, wherein said cracked solution further comprises stain remover.
9. method according to claim 8, wherein said stain remover is selected from Tween
TM20, TritonX-100
TM, Nonidet
TMP-40, Brij58
TM, Sodium desoxycholate, N-Sarkosyl L.
10. method according to claim 9, wherein said cracked solution comprises the 4M guanidine thiocyanate, 0.1M sodium acetate (pH5.0), 5%TritonX-100
TMWith the 3M urea.
11. method according to claim 1, the step of wherein said filtration cracked solution are to finish by using vacuum in the outlet of described device.
12. method according to claim 1 comprises repeating step (3) and (4).
13. method according to claim 1, wherein said cell cultures is in described cell filtration device.
14. method according to claim 13 comprises in described device and supplies air.
15. method according to claim 14, wherein said air is supplied to described culture by described hollow membrane filter.
16. from the method for cell suspension extraction DNA, this method comprises the following steps:
1) cell suspension is added in the filtration unit with hollow membrane filter;
2) if desired, cross the substratum that filters to remove the described cell that suspends;
3) add cracked solution to described cell, be incubated the sufficiently long time of described cell, so that released dna; With
4) filter the cracked solution that contains described DNA;
5) filtrate with step (4) joins in the Ion Exchange Medium;
6) with the described Ion Exchange Medium of first solution washing with material wash-out that will be except that DNA; With
7) with the described Ion Exchange Medium of second solution washing with the described DNA of wash-out.
17. method according to claim 16, wherein said Ion Exchange Medium is by diethylaminoethyl-, the inert base that QAE or quaternary ammonium group replace.
18. method according to claim 16, wherein said Ion Exchange Medium is a silicon-dioxide.
19. method according to claim 16, wherein said first solution is the alcoholic solution that does not dissolve described DNA.
20. method according to claim 16, wherein said second solution are the aqueous solution of the described DNA of dissolving.
21. method according to claim 16, wherein said filtration unit directly is attached on the device that comprises described Ion Exchange Medium.
22. from the method for microorganisms cultures extraction plasmid DNA, this method comprises the following steps:
1) microorganisms cultures that will carry plasmid is added in the filtration unit with hollow membrane filter;
2) if desired, filter to remove substratum excessively;
3) add cracked solution to described microorganism, be incubated the sufficiently long time of described microorganism, so that discharge plasmid DNA; With
4) filter the cracked solution that contains described plasmid DNA.
23. method according to claim 22, wherein said microorganisms cultures is a bacterial cultures.
24. method according to claim 22, wherein said microorganism is cultivated in described device.
25. method according to claim 22 comprises further that wherein washing is collected in the step of the cell on the hollow fiber filter in step (2).
26. method according to claim 22, wherein said cracked solution comprises the buffered soln of chaotropic agent.
27. method according to claim 26, wherein said chaotropic agent is selected from Guanidinium hydrochloride, guanidine thiocyanate, sodium iodide and sodium perchlorate.
28. method according to claim 26, wherein said cracked solution further comprises stain remover.
29. method according to claim 28, wherein said stain remover is selected from Tween
TM20, TritonX-100
TM, Nonidet
TMP-40, Brij58
TM, Sodium desoxycholate, N-Sarkosyl L.
30. method according to claim 29, wherein said cracked solution comprises the 6M Guanidinium hydrochloride, 0.75%Triton X-100
TMWith 200mM HEPES (pH6.5) or 100mMTris.HCl (pH6.4).
31. method according to claim 22, the step of wherein said filtration cracked solution are to finish by using vacuum in the outlet of described device.
32. method according to claim 22 comprises repeating step (3) and (4).
33. method according to claim 22, wherein said cell cultures is in described cell filtration device.
34. method according to claim 33 comprises in described device and supplies air.
35. method according to claim 34, wherein said air is supplied to described culture by described hollow membrane filter.
36. from the method for microorganisms cultures extraction plasmid DNA, this method comprises the following steps:
1) microorganisms cultures that will carry plasmid DNA is added in the filtration unit with hollow membrane filter;
2) if desired, filter to remove substratum excessively;
3) add cracked solution to described microorganism, be incubated the sufficiently long time of described microorganism, so that discharge plasmid DNA; With
4) filter the cracked solution that contains described plasmid DNA;
5) filtrate with step (4) joins in the Ion Exchange Medium;
6) with the described Ion Exchange Medium of first solution washing with material wash-out that will be except that plasmid DNA; With
7) with the described Ion Exchange Medium of second solution washing with the described plasmid DNA of wash-out.
37. method according to claim 36, wherein said Ion Exchange Medium are to use diethylaminoethyl-, the inert base that QAE or quaternary ammonium group replace.
38. method according to claim 36, wherein said Ion Exchange Medium is a silicon-dioxide.
39. method according to claim 36, wherein said first solution is the alcoholic solution that does not dissolve described DNA.
40. method according to claim 36, wherein said second solution are the aqueous solution of the described DNA of dissolving.
41. method according to claim 36, wherein said filtration unit directly is attached on the device that comprises described Ion Exchange Medium.
42. be used for culturing cell and therefrom extract the device of DNA, this device comprises:
Container with an opening end and a closed end, this closed end comprise the alternative hollow membrane filter of liquid that allows described internal tank and outside; With
Seal the movable sealing thing of described closed end.
43. according to the described device of claim 42, wherein said container is the cylinder with about 2 ml volumes.
44. according to the described device of claim 43, wherein said hollow membrane filter is an axially extended single cylindrical element in container.
45. according to the described device of claim 42, the wherein said sealer that is used for the closed end of tightness system can be a kind of sheet metal sealer that pulls out, or cooperates or the lid of frictional fit form seal end with screw.
46., wherein further be included in the movable sealing thing of the opening end of described container according to the described device of claim 42.
47. according to the described device of claim 46, wherein said movable sealing thing is gas-pervious lid.
48. according to the described device of claim 42, the closed end of wherein said container is suitable for fixedly comprising the filtration unit of Ion Exchange Medium.
49. according to the described device of claim 42, wherein said apparatus container is from being selected from polypropylene, polystyrene and the preparation of acrylate plastics.
50. be used for culturing cell and therefrom extract the device of DNA, this device comprises the described device of a plurality of claims 42.
51. be used for culturing cell and therefrom extract the device of DNA, this device comprises the linear combination of the described device of a plurality of claims 42, wherein each container is elongated cylinder.
52. according to the described device of claim 51, it is made of 8 containers so that corresponding with the hole of 8 * 12 hole droplet plates of standard.
53. according to the described device of claim 52, wherein said container connects by means of brittle reticulation.
54. according to the described device of claim 52, at least a portion of at least a portion of one of them wall of a container and the wall of adjacent container is adjacent.
55. according to the described device of claim 52, wherein the closed end of each container in described device combination is suitable for fixedly comprising the filtration unit of Ion Exchange Medium.
56. be used for culturing cell and therefrom extract the test kit of DNA, this test kit comprises at least a each the described device of claim 42,50 or 51 that is selected from.
57., further comprise the reagent that is used to extract described DNA according to the described test kit of claim 56.
58., further comprise the vacuum collecting tubule of the device that is suitable for engaging claim 50 or 51 according to the described test kit of claim 56.
59., further comprise the device that is used for ion-exchange purification DNA according to the described test kit of claim 56.
60. according to the described test kit of claim 59, wherein said ion exchange unit comprises the combination of the device that is suitable for engaging claim 50 or 51.
61., further comprise directly or being suitable for of being connected with the ion exchange unit of claim 60 successively engages the vacuum collecting tubule of the device of claim 50 or 51 according to the described test kit of claim 56.
62. according to the described test kit of claim 61, wherein said vacuum collecting tubule is suitable for the droplet plate in 8 * 12 holes of the standard that engages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96194606A CN1187196A (en) | 1995-06-08 | 1996-06-11 | Method and apparatus for DNA extraction |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPN3469 | 1995-06-08 | ||
| AUPO0086 | 1996-05-27 | ||
| CN96194606A CN1187196A (en) | 1995-06-08 | 1996-06-11 | Method and apparatus for DNA extraction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1187196A true CN1187196A (en) | 1998-07-08 |
Family
ID=5128735
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN96194606A Pending CN1187196A (en) | 1995-06-08 | 1996-06-11 | Method and apparatus for DNA extraction |
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| Country | Link |
|---|---|
| CN (1) | CN1187196A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100343382C (en) * | 2005-06-22 | 2007-10-17 | 殷玉和 | Multi-stage cell rupture and plasmid crude extracting reactor |
| CN1958800B (en) * | 2006-11-23 | 2010-05-12 | 广东省农业科学院兽医研究所 | Device for extracting plasmid DNA |
| CN101538567B (en) * | 2008-03-20 | 2012-09-19 | 杭州优思达生物技术有限公司 | Method for quickly processing filter-type micro nucleic acid clinical samples |
| CN101443452B (en) * | 2004-11-30 | 2013-03-27 | 梅瑞尔有限公司 | Mixing devices for chemical lysis of cells |
| CN103131628A (en) * | 2013-02-04 | 2013-06-05 | 上海交通大学 | Plant genomes deoxyribonucleic acid (DNA) quick extraction device and testing method thereof |
| CN105385676A (en) * | 2015-11-05 | 2016-03-09 | 杭州易文赛生物技术有限公司 | Method for extracting DNA, method for dissociating chromatin and application of perchlorate |
| CN106536056A (en) * | 2014-06-13 | 2017-03-22 | 儿童医学中心公司 | Products and methods to isolate mitochondria |
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1996
- 1996-06-11 CN CN96194606A patent/CN1187196A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101443452B (en) * | 2004-11-30 | 2013-03-27 | 梅瑞尔有限公司 | Mixing devices for chemical lysis of cells |
| CN100343382C (en) * | 2005-06-22 | 2007-10-17 | 殷玉和 | Multi-stage cell rupture and plasmid crude extracting reactor |
| CN1958800B (en) * | 2006-11-23 | 2010-05-12 | 广东省农业科学院兽医研究所 | Device for extracting plasmid DNA |
| CN101538567B (en) * | 2008-03-20 | 2012-09-19 | 杭州优思达生物技术有限公司 | Method for quickly processing filter-type micro nucleic acid clinical samples |
| CN103131628A (en) * | 2013-02-04 | 2013-06-05 | 上海交通大学 | Plant genomes deoxyribonucleic acid (DNA) quick extraction device and testing method thereof |
| CN106536056A (en) * | 2014-06-13 | 2017-03-22 | 儿童医学中心公司 | Products and methods to isolate mitochondria |
| CN106536056B (en) * | 2014-06-13 | 2021-07-16 | 儿童医学中心公司 | Products and methods for isolating mitochondria |
| US11491480B2 (en) | 2014-06-13 | 2022-11-08 | Children's Medical Center Corporation | Products and methods to isolate mitochondria |
| CN105385676A (en) * | 2015-11-05 | 2016-03-09 | 杭州易文赛生物技术有限公司 | Method for extracting DNA, method for dissociating chromatin and application of perchlorate |
| CN105385676B (en) * | 2015-11-05 | 2018-10-02 | 杭州易文赛生物技术有限公司 | A kind of purposes of the method and the chromatinic method of dissociation and perchlorate of extraction DNA |
| CN109932359A (en) * | 2019-02-11 | 2019-06-25 | 张丽英 | A method of utilizing clostridium botulinum in ATP bioluminescence reaction detection food |
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