CN103667253B - The method of isolating nucleic acid and device - Google Patents
The method of isolating nucleic acid and device Download PDFInfo
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- CN103667253B CN103667253B CN201210347718.9A CN201210347718A CN103667253B CN 103667253 B CN103667253 B CN 103667253B CN 201210347718 A CN201210347718 A CN 201210347718A CN 103667253 B CN103667253 B CN 103667253B
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Abstract
A kind of method of isolating nucleic acid comprises: the sample reception portion sample solution containing nucleic acid being added to silica fiber filter paper; Make buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion, thus nucleic acid is positioned according to its molecular weight difference on silica fiber filter paper.The invention still further relates to the device of relevant isolating nucleic acid.
Description
Technical field
The present invention relates to method and the device of isolating nucleic acid.
Background technology
Nucleic acid is applied to the field such as medical science and biology.Before the manipulation in vitro of nucleic acid and analysis usually need itself and other unwanted magazins' layout to come, to avoid affecting subsequent in vitro operation and analytic process.
The nucleic acid of different molecular weight provides the information important due to different reasons.Such as, thymus nucleic acid provides the important information in gene sudden change/local rearrangement congenital and day after tomorrow.Contrary with thymus nucleic acid, Yeast Nucleic Acid then reflects the biology " snapshot " of cell, because they always follow surrounding environment continue change.
In some applications, if nucleic acid can be separated according to the difference of molecular weight from single biological specimen, will significantly be simplified the collection of biological specimen and subsequent analysis.When biological specimen is very little, such as during tissue biopsy, be difficult to this biological specimen to resolve into the less biological specimen that the nucleic acid different to molecular weight carries out each self-separation further, the nucleic acid simultaneously isolating different molecular weight from same biological specimen is particularly important.
The technology of existing various different isolation/separation/purification of nucleic acid.Such as, No. 2007/104962nd, International Patent Application Publication describe a kind of by liquid sample along absorbent membrane flowing to make nucleic acid along the length distribution of film thus the method be separated from liquid sample.
No. 2009/0143570th, U.S. Patent Application Publication relates to the method for at least two kinds of cellular constituents that a kind of separation is selected from genomic deoxyribonucleic acid, Yeast Nucleic Acid and protein, and it comprises: the aqueous solution comprising cellular constituent is applied to the first solid support to adsorb genomic deoxyribonucleic acid; With comprising being applied to the second solid support by thing and adsorbing Yeast Nucleic Acid not by the Yeast Nucleic Acid that adsorbs and protein.
Increase for nucleic acid use creates the demand for quick, easy and reliable separate nucleic acid method and apparatus, therefore, is necessary method and the device of developing new isolating nucleic acid.
Summary of the invention
The object of this invention is to provide a kind of method and device of new isolating nucleic acid.
On the one hand, the method for the isolating nucleic acid that the present invention relates to comprises: the sample reception portion sample solution containing nucleic acid being added to silica fiber filter paper; Make buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion, thus nucleic acid is positioned according to its molecular weight difference on silica fiber filter paper.
On the other hand, the device of the isolating nucleic acid that the present invention relates to comprises: silica fiber filter paper, and it comprises: sample reception portion, and it receives the sample solution containing nucleic acid; And location division, its buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion after locate the nucleic acid of corresponding molecular weight.
Accompanying drawing explanation
Be described for embodiments of the invention in conjunction with the drawings, the present invention may be better understood, in the accompanying drawings:
Fig. 1 is the schematic diagram of the device provided according to the first embodiment of the present invention.
Fig. 2 is the schematic diagram of device provided according to a second embodiment of the present invention.
Fig. 3 is the schematic diagram of according to the third embodiment of the invention provided device.
Fig. 4 is the schematic diagram of device provided according to a fourth embodiment of the invention.
Fig. 5 is the electrophoretic image obtained in example 1.
Fig. 6 is the electrophoretic image obtained in example 2.
Fig. 7 is the electrophoretic image obtained in example 3.
Fig. 8 is the electrophoretic image obtained in example 4.
Fig. 9 is the electrophoretic image obtained in Fig. 3 shown device and example 5.
Figure 10 is the electrophoretic image obtained in Fig. 4 shown device and example 6.
Embodiment
Hereinafter, with reference to the accompanying drawings embodiments of the present invention will be described, well-known function and structure can not be described in detail, to avoid making because of unnecessary details the present invention become puzzling.
Unless otherwise mentioned, the ordinary meaning that the personage having general technical ability in technical term used herein or scientific terminology field belonging to the present invention understands." first ", " second ", " the 3rd " that use in patent application specification of the present invention and claims and similar word do not represent any order, quantity or importance, and are only used to distinguish different integral parts." comprise ", the similar word such as " containing " or " comprising " mean to appear at " comprising ", " containing " or " comprising " before element or object contain and appear at " comprising ", the element of " containing " or " comprising " presented hereinafter or object and equivalent, do not get rid of other elements or object." although connection ", " adjoining " or " being connected " etc. similar word be usually used in describing physics or the connection of machinery, be not defined in this, and comprise and directly or indirectly connecting.
Approximate term in specification sheets is used for modifying quantity, represents that the present invention is not limited to this concrete quantity, also comprises close to this quantity acceptable and can not cause the part of the correction of the change of relevant basic function.Accordingly, modify a numerical value with " approximately ", " about " etc., mean and the invention is not restricted to this exact numerical.In some example, approximate term may correspond to the precision of the instrument measuring numerical value.
In the following description book and claim, unless clearly pointed out in addition, single plural number is not limited.
"available" mentioned by the application, " possibility " and " can be " represent the possibility occurred under certain environment; There is the character of specifying, the possibility of feature or function; And/or by one or more ability of display, performance and be suitable for another kind of action, or the possibility relevant to the action that this is applicable to.Therefore, represent that the term modified obviously is applicable to, or can be suitable for represented ability, function for "available", " possibility " and " can be ", or purposes, consider in some cases simultaneously, the term modified may be not suitable for sometimes, can not or improper.Such as, in some cases, event or ability may be desired, and in other cases, this event or ability can not occur.This difference is contained by term "available", " possibility " and " can be ".
The present invention relates to from biological specimen nucleic acid and unwanted magazins' layout and/or from single creature sample method and apparatus disconnected from each other according to different molecular weight for nucleic acid.The method and apparatus that the present invention relates to does not need to use expensive centrifugal separation equipment and disengaging time is shorter.
In the present invention " separation " refer to from sample solution nucleic acid and unwanted magazins' layout and/or nucleic acid is disconnected from each other according to different molecular weight from single creature sample and the behavior of the isolation carried out or purification of nucleic acid or action.
In the present invention, " nucleic acid " refers to thymus nucleic acid, Yeast Nucleic Acid or its combination.Be separated the nucleic acid that obtains and can comprise the nucleic acid of single type or more than 2 dissimilar nucleic acid.Be separated the nucleic acid obtained and can be strand, double-strand, linear or ring-type.The molecular weight being separated the nucleic acid obtained also does not limit, and it in certain embodiments can several base pair (basepair, bp) in the scope of millions of bp.
" biological specimen " this term that the present invention mentions is sensu lato, and is intended to contain the various physiology or the clinical biochemical resource that comprise nucleic acid.Such Biological resources include, but not limited to global tissue, comprise biopsy material and aspirate; Cultured cell in vitro, comprises primary cell and secondary cell, transformation cell lines and histocyte and hemocyte; Body fluid, as urine, saliva, seminal fluid, secretory product, eyes cleaning and aspirate, lung cleaning and aspirate; The medium of synthesis thymus nucleic acid or Yeast Nucleic Acid; The thymus nucleic acid of chemical method or biological process synthesis or the mixture of Yeast Nucleic Acid; Fungi and plant tissue, such as leaf, root, stem and cap; Can to be present on biological specimen or in microorganism and virus; And there is the resource that maybe can exist in other any thymus nucleic acids and/or Yeast Nucleic Acid.
Sample solution can be to dissolve, suspend, mix or other modes comprise thymus nucleic acid, Yeast Nucleic Acid or its combination, or the solution of cell, cellular constituent or cell extract containing thymus nucleic acid, Yeast Nucleic Acid or its combination.Sample solution can obtain from biological specimen preparation.
Comprise use from the exemplary method of sample solution of biological specimen preparation containing nucleic acid and comprise the step that the solubilising reagent from liquid thing (chaotropicsubstances) and/or other reagent dissolves biological specimen, implementing the simplest method of this step is that biological specimen is contacted with solubilising reagent.Can be about 0.1 mol/L to about 10 mol/L from the concentration of liquid thing in solubilising reagent, such as about 1 mol/L is to about 10 mol/L.
What the present invention mentioned refers to a kind of secondary, three grades, the quaternary structure that can pass through such as to change nucleic acid from liquid thing, retains the material that the modes such as primary structure is complete cause nucleic acid unordered.Example from liquid thing includes but not limited to, any combination of Guanidinium hydrochloride, guanidinium isothiocyanate/guanidine thiocyanate, Sodium Thiocyanate 99, sodium perchlorate, sodium iodide, potassiumiodide, urea and/or these materials.Typical in a solution anion series, as follows according to the order arrangement of successively decreasing from liquid ability: CCl
3cOO
-, CNS
-, CF
3cOO
-, ClO
4 -, I
-, CH
3cOO
-, Br
-, Cl
-, CHO
2 -.
For some biological specimen, as bacterium, solubilising reagent can comprise lytic enzyme, or, precedent is being dissolved as carried out pre-treatment with lytic enzyme to this biological specimen.
In some embodiments, solubilising reagent comprises enough damping fluids.The damping fluid example used in solubilising reagent comprises three-(methylol) methylamine hydrochlorides (three-hydrochloric acid), sodium phosphate, sodium-acetate, sodium tetraborate-boric acid and Glycine-NaOH.
In some embodiments, solubilising reagent comprises reductive agent, and this reductive agent can promote from liquid thing to the sex change of rnase with help not degrade being separated of Yeast Nucleic Acid.Reductive agent example includes but not limited to, 2-aminoothyl mercaptan, three-carboxy ethyl phosphuret-(t)ed hydrogen and beta-mercaptoethanol.
In some embodiments, solubilising reagent comprise nonionogenic tenside, cats product, anion surfactant, amphoterics and or the arbitrary combination of various tensio-active agent.The example of nonionogenic tenside includes but not limited to polyoxyethylene octyl phenyl ether (Triton
tMx-100), (Octylphenoxy) poly-ethanol (IGEPAL
tMcA-630/NP-40), TRI ETHYLENE GLYCOL list lauryl alcohol (BRIJ
tM30), sorbitol anhydride laurate (SPAN
tM, or polysorbate compound family, such as polysorbate20 (i.e. TWEEN 20)
tM20), TWEEN
tM40, TWEEN
tM60 and TWEEN
tM80 (Sigma-Aldrich company, St. Louis cities).The example of cats product comprises cetrimonium bromide, dodecyltrimethyl, tetradecyl chloride base trimethyl ammonium and cetylpyridinium chloride.
In solubilising reagent tensio-active agent concentration with composition in the kind of tensio-active agent and biological specimen to be dissolved difference and change.In some embodiments, the weight concentration of tensio-active agent can be about 0.1% to about 20%.
In some embodiments, solubilising reagent comprises proteolytic enzyme, and it is at least one in such as serine protease, L-Cysteine HCL Anhydrous and metalloprotease etc.Available not containing the proteolytic enzyme of nuclease.Comprise the proteolytic enzyme as stablizers such as metal ions, also can use.After the consumption of proteolytic enzyme can be and adds, the about 0.001IU to about 10IU of every milliliter of solubilising reagent or about 0.01IU is to about 1IU.
In some embodiments, solubilising reagent is containing thymus nucleic acid lytic enzyme inhibitor and/or ribonuclease inhibitor.
In some embodiments, solubilising reagent comprises defoamer.The example of defoamer comprises containing silicon defoaming agent (as silicone oil, dimethyl polysiloxane, organic silicon emulsion, modified polyorganosiloxane and silicoorganic compound), alcohol defoaming agent (such as acetylenediol, enanthol, octanol, higher alcohols and polyoxyalkylene glycol), ethers defoamer (such as, heptyl cellosolve and nonyl cellosolve-3-heptyl Sorbitol Powder), fat and oils defoamer (as animal oil and vegetables oil), fatty acid defoamer (such as, stearic acid, oleic acid and palmitinic acid), metal soap defoamer (such as, aluminum stearate and calcium stearate), fatty acid ester defoamer (such as, natural wax and tributyl phosphate), phosphoric acid salt defoamer (such as, octylphosphonic acid sodium), amine defoamer (such as diamyl amine), amides defoamer (such as, stearic amide), other defoamers (as ferric sulfate and vanadine), with may combining arbitrarily of various defoamer.
In some embodiments, solubilising reagent comprises alcohol, and it can be primary alconol, secondary alcohol and the tertiary alcohol, as methyl alcohol, ethanol, propyl alcohol and isomers thereof and butanols and isomers etc. thereof.The weight concentration of alcohol in solubilising reagent, can be about 5% to about 90%.
In some embodiments, solubilising reagent is Illustra
tMrA1 in RNAspinMinikit (cat#25-0500-71) dissolves damping fluid, wherein adds 2-beta-mercaptoethanol (cas#60-24-2).
The method of the sample solution of preparation containing nucleic acid can by such as ultrasonication, thin projections process or the technique means such as high-speed stirring or oscillation treatment.
Please refer to the drawing 1, comprises according to the device 70 that the first embodiment of the present invention provides: long strip shape silica fiber filter paper 72, and it comprises the first afterbody 74, buffered soln loading portion 76, sample reception portion 78 and second afterbody 79 contrary with the first afterbody 74.Buffered soln loading portion 76 relative to the second afterbody 79 closer to the first afterbody 74.Sample reception portion 78 than buffered soln loading portion 76 closer to the second afterbody 79.
With reference to Fig. 2, the collecting pad 3 that device 1 provided according to a second embodiment of the present invention comprises sample pad 2 and is connected in the longitudinal direction with sample pad 2.Sample pad 2 is the silica fiber filter paper similar to silica fiber filter paper 72 in Fig. 1, and it comprises the first afterbody 5, sample reception portion 4 and second afterbody (unmarked) contrary with the first afterbody 5.Collecting pad 3 can be silica fiber filter paper or stops pad, and it is connected with the second afterbody of sample pad 2.
With reference to Fig. 3, the imbibition pad 13 that according to the third embodiment of the invention provided device 10 comprises sample pad 12 and is connected in the longitudinal direction with sample pad 12.Sample pad 12 is the silica fiber filter paper similar to silica fiber filter paper 72 in Fig. 1, and it comprises the first afterbody 15, buffered soln loading portion 17, sample reception portion 14 and second afterbody 16 contrary with the first afterbody 15.Buffered soln loading portion 17 relative to the second afterbody 16 closer to the first afterbody 15.Sample reception portion 14 than buffered soln loading portion 17 closer to the second afterbody 16.
With reference to Fig. 4, device 100 provided according to a fourth embodiment of the invention comprises sample pad 170, the termination pad 180 that is connected in the longitudinal direction with sample pad 170 and with the imbibition pad 130 stopping pad 180 and be connected in the longitudinal direction.Sample pad 170 is the silica fiber filter paper similar to silica fiber filter paper 72 in Fig. 1, and it comprises the first afterbody 150, buffered soln loading portion 120, sample reception portion 140 and second afterbody 160 contrary with the first afterbody 150.Buffered soln loading portion 120 relative to the second afterbody 160 closer to the first afterbody 150.Sample reception portion 140 than buffered soln loading portion 120 closer to the second afterbody 160.
Silica fiber filter paper 72,2,12,170 can be any sorption sample solution and does not suppress the storage of nucleic acid or the porous imbibition silica fiber filter paper of subsequent analysis.In some embodiments, silica fiber filter paper 72,2,12,170 is made up of the pure silica fiber of binder free.In some embodiments, it is about 98% by efficiency that the particle that silica fiber filter paper 72,2,12,170 pairs of granular sizes are not less than 2.2 microns stops, and basis weight is about 85g/m2, and thickness range is about 300 microns to about 600 microns.Can be applicable to the example of silica fiber filter paper of the present invention, include but not limited to can to buy from New Jersey General Electric medical company
gradeQM-A quartz microfibre filter paper and the AQFA silica fiber filter paper can buied from Massachusetts, United States Millipore company.
Sample solution containing nucleic acid is applied to the sample reception portion 78,4,14,140 of silica fiber filter paper 72,2,12,170.Sample reception portion 78,4,14,140 can be any shape or structure that can receive sample solution.In some embodiments, the sample reception portion 78,4,14,140 of silica fiber filter paper 72,2,12,170 comprises solubilising reagent, and biological specimen then can directly apply to sample reception portion 78,4,14,140.In these cases, biological specimen is exactly the sample solution containing nucleic acid.
Buffered soln can be any solvent of nucleic acid.In some embodiments, buffered soln is three (methylol) aminomethane ethylenediamine tetraacetic acid (EDTA) (TE) buffered soln, and replaces the phosphate buffer soln after three (methylol) aminomethane and at least one in TE buffered soln by hydroxyethyl piperazine second thiosulfonic acid (HEPES).
Buffered soln spreads along silica fiber filter paper 72,2,12,170 length direction and passes through sample reception portion 78,4,14,140.Buffered soln loading portion can be any shape or structure that can receive buffered soln.In some embodiments, buffered soln is applied to buffered soln loading portion 76,17,120.In some embodiments, the first afterbody 5 is dipped in buffered soln, thus buffered soln spreads from the first afterbody 5, and in the case, the first afterbody 5 is exactly buffered soln loading portion.
Stopping pad 3,180 is elongate strip made by the material being different from silica fiber filter paper.In some embodiments, stop pad 3,180 be made up of Mierocrystalline cellulose, as
grade31ET paper.
Imbibition pad 13,130 is obtained by imbibition material, as Mierocrystalline cellulose, silicon-dioxide microfibre filter paper and glass fibre.In some embodiments, imbibition pad 13,130 is provided by General Electric's medical company of New Jersey
obtained by grade470 special purpose filter paper.
As can be seen from aftermentioned experimental example, buffered soln along silica fiber filter paper 72,2,12,170 length direction diffusion, by sample reception portion 78,4,14,140 after, nucleic acid is located according to the difference of molecular weight on the fine micro-filtration paper 72,2,12,170 of quartz.Specifically, compared with the nucleic acid that the nucleic acid that molecular weight is high is low with molecular weight, its be positioned distance sample reception portion 78,4,14,140 nearer places.
In some embodiments, the nucleic acid that major part has the first molecular weight is located near sample reception portion 78,4,14,140.In this case, sample reception portion 78,4,14,140 is location divisions of the nucleic acid with the first molecular weight.In some embodiments, the first molecular weight refer at least about 50,000 base pairs (50kilobasepairs, 50kb) or from about 50kb to the scope of about 150kb in.In some embodiments, about 50kb refers to the scope of 50kb ± 15kb.
In some embodiments, the nucleic acid that major part has the second molecular weight is located near the second afterbody 79,6,16,160.In this case, the second afterbody 79,6,16,160 is location divisions of the nucleic acid with the second molecular weight.In some embodiments, the second molecular weight is in the scope being less than about 50kb.
In some embodiments, stop pad 180,3(when collecting pad 3 is for stopping padding) and silica fiber filter paper 170,2 adjacent, buffered soln along the diffusion of silica fiber filter paper 170,2 length direction, by sample reception portion 140,4 after, the interface that major part is positioned in sample pad 170,2 with the nucleic acid of buffered soln migration and stops between pad 180,3.
In some embodiments, nucleic acid is positioned between sample reception portion 78,4,14,140 and second afterbody 79,6,16,160, thus the position point of these nucleic acid is between sample reception portion 78,4,14,140 and second afterbody 79,6,16,160.
In some embodiments, before buffered soln diffusion, make scavenging solution along the diffusion of silica fiber filter paper 72,2,12,170 length direction, by sample reception portion 78,4,14,140.Scavenging solution is not having external force only by spreading on silica fiber filter paper in capillary situation, taking away the impurity lower than nucleic acid of sorptive power on silica fiber filter paper, to be separated with impurity phase unwanted in sample solution by nucleic acid.
Sample solution containing nucleic acid, scavenging solution and buffered soln can with pipe, transfer pipet, be automatically injected device or any other be suitable for method or instrument add on silica fiber filter paper.
Scavenging solution can comprise the enzyme that can decompose impurity (such as protein).In addition, it can comprise deoxyribonuclease, rnase or analogue as required.The use comprising the scavenging solution of deoxyribonuclease can contribute to the selective recovery of Yeast Nucleic Acid.Equally, the scavenging solution comprising rnase can contribute to optionally reclaiming thymus nucleic acid.
Scavenging solution can comprise water-miscible organic solvent and/or water-soluble salt.Scavenging solution to wash out in sample solution the impurity of sorption on silica fibre filter paper together with nucleic acid.Therefore, it can allow nucleic acid keep the composition of sorption containing by impurity while silica fiber filter paper desorption.The water-miscible organic solvent of nucleic acid indissoluble, as alcohols, is applicable to from other compositions beyond silica fiber filter paper desorb nucleic acid.Meanwhile, water-soluble salt include the absorption that can strengthen nucleic acid in, assist the selectivity desorb to unnecessary impurity.
The example of the water-miscible organic solvent in scavenging solution comprises methyl alcohol, ethanol, Virahol, n-propyl alcohol, propyl carbinol and acetone, and wherein ethanol is comparatively suitable.The weight concentration of water-miscible organic solvent in scavenging solution can be about 20% to 100% or about 40% to about 100%.
Water-soluble salt in scavenging solution can be halide salt or three (methylol) aminomethane.
In some embodiments, scavenging solution is the TE buffered soln containing 80% ethanol.
Scavenging solution can be identical at the point of addition of silica fiber filter paper with the sample solution containing nucleic acid, namely all adds in sample reception portion, or identical at the point of addition of silica fiber filter paper with buffered soln, namely all adds in buffered soln loading portion.Scavenging solution also can be different from the position interpolation in sample reception portion and buffered soln loading portion from silica fiber filter paper.
In some embodiments, scavenging solution diffuses to the second afterbody 79,6,16,160, impurity band to the second afterbody 79,6,16,160 unwanted in sample solution.The second afterbody 79,6,16,160 was excised again before buffered soln diffusion.In this way, the nucleic acid be positioned on residual quartz fiber filter paper be unwanted impurity is purified/is separated/isolation after nucleic acid.
In some embodiments, at scavenging solution by sample reception portion 4, along silica fiber filter paper 2 length direction, stop pad, imbibition pad or silica fiber filter paper towards collecting pad 3() and after the second afterbody 6 of device 1 spreads, change collecting pad 3, thus make buffered soln diffuse to the collecting pad 3 of replacing from sample pad 2.
Silica fiber filter paper 72,1,12,170, collecting pad 3, imbibition pad 13,130, stopping pad 180 can by such as polyester
or the back lining materials of the plastic material such as polyethylene terephthalate (PET) and so on provides support.
In some embodiments, device 70,1,10,100 comprises plastic casing (not shown), it is by silica fiber filter paper 72,1,12,170, collecting pad 3, imbibition pad 13,130, stop pad 180 in the inner coated, but by corresponding aperture, sample reception portion 78,4,14,140 and/or buffered soln loading portion 76,5,17,120 are exposed, thus make the sample solution containing nucleic acid, scavenging solution and buffered soln can be added to sample reception portion 78,4,14,140 and/or buffered soln loading portion 76,5,17,120.In some embodiments, sample reception portion 78,4,14,140 and/or buffered soln loading portion 76,5,17,120 can be positioned at outside housing.
Device 70,1,10,100 of the present invention can be simple nucleic acid purification, a separating device, also can be the integral part of whole foranalysis of nucleic acids equipment/Instrument assembly.
After buffered soln diffusion, the nucleic acid be positioned on silica fiber filter paper can be carried out the subsequent disposal such as elution in low ionic strength or with under the condition of water.
In the present invention " sorption " refer to that sample solution is by absorption and sorption or mix or mix sample reception portion, makes to be not easy to remove from sample reception portion, unless had a mind to or accidentally reached the condition of the composition removing sorption from sample reception portion.
In the present invention " major part " refer to reach total amount at least about 15%, at least about 50% or at least about 90%.For example, " nucleic acid that major part has the first molecular weight is positioned near sample reception portion " refers to have the first molecular weight and the nucleic acid being positioned sample reception portion, can be the nucleic acid in the sample solution being attracted in sample reception portion with the first molecular weight at least about 15%, at least about 50% or at least about 90%.Similarly, " nucleic acid that major part has the second molecular weight is positioned near the second afterbody " refers to have the second molecular weight and the nucleic acid being positioned the second afterbody, can be the nucleic acid in the sample solution being attracted in sample reception portion with the second molecular weight at least about 15%, at least about 50% or at least about 90%.
The intrinsic capillary force of the porousness of silica fiber filter paper is as making scavenging solution and/or buffered soln along the diffusion of silica fiber filter paper, power by sample reception portion.Imbibition pad 13,130 relies on its powerful wicking action gravitational attraction scavenging solution and/or buffered soln to spread towards absorption layer 13,130.Different for the sorptive force of cellular quartz fiber filter paper from nucleic acid based on impurity, impurity taken away by scavenging solution and/or buffered soln, nucleic acid is fully separated with impurity in sample solution, eliminate producing the instrument of external impetus (as centrifugal force and pressure) and there is the demand of the talent of certain skills, make to be separated into possibility at the scene of remote districts.
Buffered soln spreads along silica fiber filter paper length direction, by sample reception portion, nucleic acid is moved on silica fiber filter paper, and molecular weight is lower, and the distance that nucleic acid moves from sample reception portion is far away.When buffered soln is diffused into by when being different from termination pad or imbibition pad that silica fiber filter paper material makes from silica fiber filter paper, the nucleic acid along with buffered soln migration will stop and the interface being positioned silica fiber filter paper and stopping between pad or imbibition pad.Therefore, spread by buffered soln, nucleic acid is positioned at the corresponding position of silica fiber filter paper with molecular size range, the object of isolating nucleic acid and impurity and/or different molecular weight nucleic acid can be reached.
Experimental example
Following experimental example for those skilled in the art implement the invention provides further guidance.Example does not limit the scope of the present invention defined in claims.
Example 1 thymus nucleic acid dimension analysis
Mouse genome thymus nucleic acid solution (5ul, health from BeiJing, China is century bio tech ltd) and 5ul human genome thymus nucleic acid solution (ClontechLaboratories, Inc. from California, USA) be placed in two be separated containing the sample cells of 1% agarose gel to carry out pulsed field gel electrophoresis.
Pulsed field gel electrophoresis is by the CHEF in Bio-rad laboratory
xA system voltage be 6v/cm and temperature be 10 ° of C environment in carry out 24 hours.After electrophoresis, agarose gel, by 1xSYBRGreenI solution-dyed 5 hours, uses the Typhoon that New Jersey General Electric medical company provides afterwards
tMtrio
tMmulti-modal imaging instrument develops.
Pulsed field gel electrophoresis image as shown in Figure 5.In Figure 5, swimming lane I, III characterize MidRangeIIPFG mark, and swimming lane II characterizes 5ul mouse genome thymus nucleic acid solution, and swimming lane IV characterizes 5ul human genome thymus nucleic acid solution.
As can be seen from Figure 5, the molecular weight of mouse genome thymus nucleic acid is less than about 50kb, and human genome thymus nucleic acid is at least about 50kb and in the scope of about 50kb to about 150kb.
Example 2 lower molecular weight (< 50kb) thymus nucleic acid
Please refer to Fig. 2, the wide 4mm of device 1(, long 5.5cm) comprise the wide 4mm of sample pad 2(, long 2.5cm) and have the wide 4mm of collecting pad 3(of 5mm overlap length, long 3.5cm with sample pad 2).Sample pad 2 and collecting pad 3 all obtain from New Jersey General Electric medical company
gradeQM-A quartz microfibre filter paper.There is 1.5cm in the sample reception portion 4 of sample pad 2 apart from the first afterbody 5 of sample pad 2.
Mouse genome thymus nucleic acid sample solution (2.5ul, 100ng/ul, molecular weight < 50kb, with used in example 1) adds in the micro-centrifuge tube of a 1.5ml, then with 2.5ul damping fluid (66.87% Guanidinium hydrochloride, 4%Triton
tMx-100) mix.
Before the sample reception portion 4 mixture being transferred to sample pad 2, first this micro-centrifuge tube is rocked about 15 seconds.
After 5 seconds, sample mixture is absorbed, first afterbody 5 of sample pad 2 is placed in containing in a groove of 150ul scavenging solution (the TE buffered soln (10mMTrispH8,0.1mMEDTA) containing 80% ethanol) along device length direction diffusion scavenging solution of 96 frids.
After 5 minutes, device 1 is removed, and sample pad 2 and collecting pad 3 are separated, and is in the baking oven of 37 ° of C dry 10 minutes in temperature.After drying, sample pad 2 is according to above-mentioned same way and new fitting together with the identical collecting pad 3 crossed.
The groove containing 150ulTE buffered soln (10mMTrispH8,0.1mMEDTA) that first afterbody 5 is placed in 96 frids is used for along device 1 to spread TE buffered soln.
After 5 minutes, device 1 is removed, and is in the baking oven of 37 ° of C dry 30 minutes in temperature.
After drying, device 1 is cut into the small pieces of wide 4mm, long 5mm.Each small pieces is respectively placed in the sample cell containing 0.8% agarose gel.
It is electrophoresis 45 minutes under the condition of 120V at voltage.After electrophoresis, to dye agarose gel 1.5 hours with 1xSYBRGreenI, use Typhoon afterwards
tMtrio
tMmulti-modal imaging instrument develops.
Electrophoretic image as shown in FIG. 6, in figure, the 1 to 12 swimming lane characterizes the small pieces of the first afterbody 5 to the second afterbody 6 arranged according to position in the device 1, specifically, 1 to 5 swimming lane characterizes the small pieces from sample pad 2,6 to 12 swimming lane then characterizes the small pieces from collecting pad 3,13rd swimming lane is blank, and the 14th swimming lane characterizes thymus nucleic acid mark (Japanese TakaraDL15000).This image shows, and after scavenging solution and the diffusion of TE buffered soln, most of mouse genome thymus nucleic acid (molecular weight < 50kb) is positioned at the second afterbody 6 neighbouring (the 11st, 12 swimming lanes) of device 1.In other words, the second afterbody 6 is the location divisions in this example.
Example 3 high molecular (>=50kb) thymus nucleic acid
Repeat the experiment of example 2, but replace the mouse genome thymus nucleic acid in example 2 with human genome thymus nucleic acid (molecular weight >=50kb, with used same in example 1).
Electrophoretic image as shown in FIG. 7, in figure, the 1 to 12 swimming lane characterizes the small pieces of the first afterbody 5 to the second afterbody 6 arranged according to position in the device 1,1 to 5 swimming lane is the small pieces from sample pad 2,6 to 12 swimming lane is then the small pieces from collecting pad 3, and the 13rd swimming lane characterizes thymus nucleic acid mark (Japanese TakaraDL15000).
Image shows, and after scavenging solution and the diffusion of TE buffered soln, most of human genome thymus nucleic acid is still retained in sample reception portion 4(the 3rd, 4 swimming lanes) near.In other words, most of human genome thymus nucleic acid is positioned sample reception portion 4, and sample reception portion 4 is the location divisions in this example.
Example 4 high molecular thymus nucleic acid and lower molecular weight thymus nucleic acid
Human genome thymus nucleic acid sample solution (2.5ul, 100ng/ul, with used in example 1) add in the micro-centrifuge tube of a 1.5ml, then (Japanese TakaraDL15000 is marked with 2.5ul thymus nucleic acid, comprise 7 molecular weight: 15kb, 10kb, 7.5kb, 5kb, 2.5kb, 1kb, 250bp) and 5ul damping fluid (66.87% Guanidinium hydrochloride, 4%Triton
tMx-100) mix.
The identical mode process of mixture in same example 2, but collecting pad 3 is 4cm length, and scavenging solution and TE buffered soln are all the amounts of twice in example 2.
As shown in FIG. 8, in figure, the 1st swimming lane characterizes thymus nucleic acid mark (Japanese TakaraDL15000) to electrophoretic image, and the 2 to 14 swimming lane characterization apparatus 1 is from the small pieces of the first afterbody 5 to the second afterbody 6.
This figure shows, scavenging solution and TE buffered soln diffusion after, at sample reception portion 4(the 4th swimming lane) near only have human genome thymus nucleic acid, and, at the second afterbody 6(the 14th swimming lane) only have low-molecular-weight thymus nucleic acid to mark.That is, for human genome thymus nucleic acid, location division is sample reception portion 4, low-molecular-weight thymus nucleic acid is marked, second afterbody 6 is its location divisions, and for molecular weight between being positioned the nucleic acid between the nucleic acid molecular weight in sample reception portion 4 and the nucleic acid molecular weight being positioned the second afterbody 6, its location division is between the second afterbody 6 and sample reception portion 4.
Example 5 thymus nucleic acid and Yeast Nucleic Acid
Please refer to Fig. 9, use the device 10 in Fig. 3, it comprises a wide 4mm of sample pad 12(, long 6cm's
gradeQM-A quartz microfibre filter paper), one and sample pad 12 partly overlapping imbibition pad 13(
grade470 special purpose filter paper) and one by sample pad 12 and the coated housing of imbibition pad 13 (not shown, the A-10 plastic casing of an outstanding bio tech ltd, Shanghai of Chinese Shanghai).
Comprise 1.2x10
6the fusion tissue culture (stem cell culture) of cell is rotated rapidly and is removed floating layer medium.The Illustra of cell pellets and 35ul
tMthe RA1 of RNAspinMinikit (cat#25-0500-71) dissolves 2-beta-mercaptoethanol (BME, cas#60-24-2) the rotation mixing of damping fluid and 0.35ul.
By an opening (not shown) of housing, 5ul cytolysate is added to and the sample reception portion 14 of the first afterbody 15 of sample pad 12 apart from 2.7cm.After drying, the totalJurkatRNA (1ug/ul) of 1ul adds to sample reception portion 14 by this opening.
Not containing the TE buffered soln (150ul of deoxyribonuclease/rnase, 10mMTrispH8,0.1mMEDTA) to add to the first afterbody 15 of sample pad 12 apart from the buffered soln loading portion 17 for 1cm by another opening (not shown) of housing, spread along device 10 length direction.
After 10 minutes, sample pad 12 is taken out from housing and is placed in the agarose gel of 0.8%.Electrophoresis is carried out 45 minutes at the voltage of 110V.Afterwards, with insertion dyestuff, agarose gel is dyeed 1 hour, re-use Typhoon
tMtrio
tMmulti-modal imaging instrument develops.
The result display of nucleic acid location in fig .9, according to the positional alignment corresponding to sample pad 12 shown in figure, but that swimming lane A shows is NEB1kbladder, swimming lane B is the development of cytolysate and the identical check sample of totalJurkatRNA consumption received with sample reception portion 14, for making comparisons, without TE buffered soln DIFFUSION TREATMENT before this sample electrophoresis.
As can be seen from Figure 9, at TE buffered soln along after device 10 spreads, the thymus nucleic acid (DNA) of high molecular is positioned near sample reception portion 14, and Yeast Nucleic Acid (RNA) components migrate be positioned to the second afterbody 16.That is, sample reception portion 14 is the location division of high molecular thymus nucleic acid and the second afterbody 16 is location divisions of Yeast Nucleic Acid composition.
Example 6 thymus nucleic acid and Yeast Nucleic Acid
Please refer to Figure 10, the device 100 in application drawing 4, it comprises a wide 4mm of sample pad 170(, long 4cm's
gradeQM-A quartz microfibre filter paper), one is padded the wide 4mm of 180(with partly overlapping termination of sample pad 170, long 2.5cm's
grade31ET paper), one and termination pad 180 partly overlapping imbibition pad 130(
grade470 special purpose filter paper) and one by coated for aforementioned pad housing (not shown, the A-10 plastic casing of an outstanding bio tech ltd, Shanghai of Chinese Shanghai).
The fusion tissue culture (stem cell culture) comprising 1.2x106 cell is rotated rapidly and is removed floating layer medium.The Illustra of cell pellets and 35ul
tMthe RA1 of RNAspinMinikit (cat#25-0500-71) dissolves 2-beta-mercaptoethanol (BME, cas#60-24-2) the rotation mixing of damping fluid and 0.35ul.
By an opening (not shown) of housing, 5ul cytolysate is added to and the sample reception portion 140 of the first afterbody 150 of sample pad 170 apart from 2.7cm.After drying, the totalJurkatRNA (1ug/ul) of 1ul adds to sample reception portion 140 by this opening.
150ul is not containing the TE buffered soln (10mMTrispH8 of deoxyribonuclease/rnase, 0.1mMEDTA) to add to the first afterbody 150 of sample pad 170 apart from the buffered soln loading portion 120 for 1cm by another opening (not shown) of housing, spread along device 100 length direction.
After 10 minutes, sample pad 170 and termination pad 180 take out from housing and are placed in the agarose gel of 0.8%.Electrophoresis is carried out 45 minutes at the voltage of 110V.Afterwards, with insertion dyestuff, agarose gel is dyeed 1 hour, re-use Typhoon
tMtrio
tMmulti-modal imaging instrument develops.
The result display of nucleic acid location in Fig. 10, according to sample pad 170 with stop the corresponding positional alignment of pad 180 shown in figure, but that swimming lane D shows is NEB1kbladder, swimming lane C is the development of cytolysate and the identical check sample of totalJurkatRNA consumption received with sample reception portion 140, for making comparisons, without TE buffered soln DIFFUSION TREATMENT before this sample electrophoresis.
As can be seen from Figure 10, thymus nucleic acid (DNA) is positioned near sample reception portion 140, and at the second afterbody 160 of sample pad 170 and the concentration degree highly significant of Yeast Nucleic Acid (RNA) on stopping between pad 180 interface.In other words, the location division of to be the location division of thymus nucleic acid and the second afterbody 160 and the interface stopped between pad 180 be in sample reception portion 140 Yeast Nucleic Acid.
Although be described in detail Partial Feature of the present invention and described in a specific embodiment, under the prerequisite not departing from spirit of the present invention, various change and replacement can be carried out to the present invention.Same, those skilled in the art also can obtain disclosed by the invention other according to normal experiment and change and equivalent.All these change, and replace and within the design of claim that equivalent all defines in the present invention and scope.
Claims (16)
1. a method for isolating nucleic acid, is characterized in that, comprising:
Sample solution containing nucleic acid is added to the sample reception portion of silica fiber filter paper, silica fiber filter paper is elongated, comprise the first afterbody, buffered soln loading portion, sample reception portion and the second afterbody, buffered soln loading portion relative to the second afterbody closer to the first afterbody, sample reception portion than buffered soln loading portion closer to the second afterbody;
Make buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion, thus nucleic acid is positioned according to its molecular weight difference on silica fiber filter paper.
2. the method for isolating nucleic acid as claimed in claim 1, it is characterized in that, it comprises, and provides the imbibition pad that adjacent with the second afterbody.
3. the method for isolating nucleic acid as claimed in claim 2, it is characterized in that, it comprises, and making buffered soln along before the diffusion of silica fiber filter paper length direction, makes cleaning solution diffuse to imbibition pad along silica fiber filter paper length direction.
4. the method for isolating nucleic acid as claimed in claim 1, it is characterized in that, buffered soln loading portion is the first afterbody, and is immersed in buffered soln.
5. the method for isolating nucleic acid as claimed in claim 1, it is characterized in that, it comprises, provide one adjacent with the second afterbody, by the termination pad be different from made by silica fiber filter paper material.
6. the method for isolating nucleic acid as claimed in claim 1, it is characterized in that, it comprises, make buffered soln along silica fiber filter paper length direction diffusion before, make scavenging solution diffuse to the second afterbody along silica fiber filter paper length direction and excise the second afterbody.
7. the method for isolating nucleic acid as claimed in claim 1, is characterized in that, nucleic acid is thymus nucleic acid, Yeast Nucleic Acid or its combination.
8. the method for isolating nucleic acid as claimed in claim 1, it is characterized in that, buffered soln is the solvent of nucleic acid.
9. the method for isolating nucleic acid as claimed in claim 1, it is characterized in that, it comprises: making buffered soln along before the diffusion of silica fiber filter paper length direction, make scavenging solution along the diffusion of silica fiber filter paper length direction, by sample reception portion.
10. a device for isolating nucleic acid, is characterized in that, it comprises:
Silica fiber filter paper, it is elongated, comprise: the first afterbody, buffered soln loading portion, sample reception portion, the second afterbody and location division, buffered soln loading portion relative to the second afterbody closer to the first afterbody, sample reception portion than buffered soln loading portion closer to the second afterbody, sample reception portion receives containing the sample solution of nucleic acid, location division buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion after locate the nucleic acid of corresponding molecular weight; And
Adjacent with the second afterbody, by the termination pad be different from made by silica fiber filter paper material.
The device of 11. isolating nucleic acids as claimed in claim 10, it is characterized in that, location division is sample reception portion.
The device of 12. isolating nucleic acids as claimed in claim 10, it is characterized in that, it comprises, the housing of coated silica fiber filter paper.
The device of 13. 1 kinds of isolating nucleic acids, is characterized in that, it comprises:
Silica fiber filter paper, it is elongated, comprise: the first afterbody, buffered soln loading portion, sample reception portion, the second afterbody and location division, buffered soln loading portion is the first afterbody, sample reception portion than buffered soln loading portion closer to the second afterbody, sample reception portion receives containing the sample solution of nucleic acid, location division buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion after locate the nucleic acid of corresponding molecular weight.
The device of 14. 1 kinds of isolating nucleic acids, is characterized in that, it comprises:
Silica fiber filter paper, it is elongated, comprise: the first afterbody, buffered soln loading portion, sample reception portion, the second afterbody and location division, buffered soln loading portion relative to the second afterbody closer to the first afterbody, sample reception portion than buffered soln loading portion closer to the second afterbody, sample reception portion receives containing the sample solution of nucleic acid, location division buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion after locate the nucleic acid of corresponding molecular weight; And
The imbibition pad adjacent with the second afterbody.
The device of 15. 1 kinds of isolating nucleic acids, is characterized in that, it comprises:
Silica fiber filter paper, it is elongated, comprise: the first afterbody, buffered soln loading portion, sample reception portion, location division and the second afterbody, buffered soln loading portion relative to the second afterbody closer to the first afterbody, sample reception portion than buffered soln loading portion closer to the second afterbody, sample reception portion receives the sample solution containing nucleic acid, location division buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion after locate the nucleic acid of corresponding molecular weight, location division is the second afterbody.
The device of 16. 1 kinds of isolating nucleic acids, is characterized in that, it comprises:
Silica fiber filter paper, it is elongated, comprise: the first afterbody, buffered soln loading portion, sample reception portion, location division and the second afterbody, buffered soln loading portion relative to the second afterbody closer to the first afterbody, sample reception portion than buffered soln loading portion closer to the second afterbody, sample reception portion receives the sample solution containing nucleic acid, location division buffered soln along the diffusion of silica fiber filter paper length direction, by sample reception portion after locate the nucleic acid of corresponding molecular weight, position point is between sample reception portion and the second afterbody.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1665939A (en) * | 2002-07-04 | 2005-09-07 | 普里马根控股公司 | Storing and detecting nucleic acid administered to a solid carrier |
| US20050214926A1 (en) * | 2004-02-20 | 2005-09-29 | Ralf Zielenski | Adsorption of nucleic acids to a solid phase |
| CN1749264A (en) * | 2004-09-17 | 2006-03-22 | 株式会社日立高新技术 | Method of extracting nucleic acid method of isolation same |
| CN101443456A (en) * | 2006-03-11 | 2009-05-27 | 英国环境食物及农村事务部中央科学实验室 | Purification method and kits |
| CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1665939A (en) * | 2002-07-04 | 2005-09-07 | 普里马根控股公司 | Storing and detecting nucleic acid administered to a solid carrier |
| US20050214926A1 (en) * | 2004-02-20 | 2005-09-29 | Ralf Zielenski | Adsorption of nucleic acids to a solid phase |
| CN1749264A (en) * | 2004-09-17 | 2006-03-22 | 株式会社日立高新技术 | Method of extracting nucleic acid method of isolation same |
| CN101443456A (en) * | 2006-03-11 | 2009-05-27 | 英国环境食物及农村事务部中央科学实验室 | Purification method and kits |
| CN102154498A (en) * | 2011-03-21 | 2011-08-17 | 厦门大学 | Nucleic acid detecting method |
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