CN102115739A - Method, reagent and kit for separating nucleic acids - Google Patents
Method, reagent and kit for separating nucleic acids Download PDFInfo
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- CN102115739A CN102115739A CN2009102171687A CN200910217168A CN102115739A CN 102115739 A CN102115739 A CN 102115739A CN 2009102171687 A CN2009102171687 A CN 2009102171687A CN 200910217168 A CN200910217168 A CN 200910217168A CN 102115739 A CN102115739 A CN 102115739A
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Abstract
The invention provides a simple and quick method for separating nucleic acids, which comprises the following steps: providing a nucleic acid separation reagent, mixing the nucleic acid separation reagent with a biological sample containing one or multiple nucleic acids, and separating the nucleic acids from the biological sample by one step under a proper reaction condition without pretreating the sample. The reagent contains 1-40 wt% of polyethylene glycol (PEG), and/or higher than 30 wt% of low-molecular-weight alcohol, salt and surfactant. The nucleic acids can be combined with a solid-phase vector by changing the solubility of the nucleic acids, thereby separating the nucleic acids. In addition, the invention also provides a reagent and kit for separating nucleic acids.
Description
Technical field
The invention relates to a kind of method of isolating nucleic acid, and be particularly to a kind of separate nucleic acid method and separate nucleic acid reagent of one step.
Background technology
In recent years, rises such as medical science and criminal evaluation are learned, translated to molecular diagnosis, medicine genosome, and the demand of various foranalysis of nucleic acids also increases rapidly, yet, no matter carry out which kind of foranalysis of nucleic acids, at first necessarily require highly purified nucleic acid, and separate nucleic acid method easily and fast.
When analyzing, because the time that test substance exists in liquid sample is very of short duration, and the impurity in the sample can influence the accuracy that is detecting, therefore develop the isolation technique that a kind of nucleic acid, promptly utilize a solid phase carrier bind nucleic acid, or with the tubing string inwall as solid phase carrier, add microballon again to tubing string, again microballon is separated after utilizing the microballon bind nucleic acid.In addition, also developing the method that to utilize the magnetic bead isolating nucleic acid at present, mainly is to utilize the magnetic bead bind nucleic acid, utilizes magnetic that the magnetic bead of bind nucleic acid is separated again.
Along with the demand of separate nucleic acid is more and more frequent, the sample of action required source is also more and more extensive, because magnetic separation technique does not need centrifugal or step such as vacuumizes, therefore can save treatment time and cost.In addition, the purge process of magnetic resolution is quick, easy to operate, therefore magnetic resolution develops into main separate nucleic acid method gradually at present.
Common on the market separate nucleic acid technology mainly is to handle complex samples earlier nucleic acid is disengaged at present, utilize solid phase in conjunction with isolating nucleic acid again, the Chaotropic Salt (chaotropic salt) that the solid phase separate part is used Boom Patent (No. 5234809 patent of the U.S.) is adsorbed to the nucleic acid in the sample on the silicon materials (Silica), through after the cleaning step, re-use to dash and carry (elution, wash-out) damping fluid washes the nucleic acid on the silicon materials get off, and reaches the effect of isolating nucleic acid.In addition, No. 6274386 patent of the U.S. then is to use the magnetic solid phase carrier absorption nucleic acid of siliceous surfacing.
Yet anyway, all separate nucleic acid methods all need be through dissolving (1ysis), extraction, precipitation, clean, dash and 5 steps such as put forward, and the time that it spent is long and pollute easily.Therefore, the invention provides fast and easily a separate nucleic acid method and a reagent (composition), can save the tradition rapid separate nucleic acid method of multistep complicated and consuming time.
Summary of the invention
The present invention provides a kind of method of isolating nucleic acid, after this method comprises that the biological specimen that a separation agent and is contained nucleic acid mixes, described separation agent can make nucleic acid separate and be bonded on the solid phase carrier in biological sample, do not need the sample pre-treatment promptly can the single stage isolating nucleic acid, it is characterized in that: the separation agent of use comprises the polyoxyethylene glycol of 1～40wt% (PEG) and/or greater than lower molecular weight alcohols, salt and the tensio-active agent of 30wt%, is bonded to solid phase carrier by changing nucleic acid solubleness.
The present invention provides a kind of reagent of isolating nucleic acid in addition, and this reagent comprises the polyoxyethylene glycol (PEG) of 1～40wt% and/or greater than lower molecular weight alcohols, a salt, a tensio-active agent and the solid phase carrier of 30wt%.
The present invention also provides a kind of cover group of isolating nucleic acid, this cover group comprises a separation agent, it contains the polyoxyethylene glycol (PEG) of 1～40wt% and/or greater than lower molecular weight alcohols, salt, tensio-active agent, solid phase carrier and/or the proteolytic enzyme of 30wt%, and a container.
Preferable, among the present invention:
The molecular weight of described lower molecular weight alcohols is 30～100g/mol.
Described lower molecular weight alcohols is an ethanol.
The weight concentration of described lower molecular weight alcohols is 40～60%.
The weight concentration of described polyoxyethylene glycol is 5～20%.
Described solid phase carrier comprises metal, metal oxide, silicide or polymkeric substance.
Described solid phase carrier comprises magnetic substance.
Described salt comprises alkali metal group, alkaline earths and/or ammonium salt halogenide.
Described tensio-active agent comprises Tween-20, Triton X-100, NP40, Brij35 or CHAPS.
Described reagent also comprises a proteolytic enzyme.
Described reagent also comprises a carbohydrate.Wherein said reagent comprises a polyose or polysaccharide derivatives.Wherein said polyose comprises glycogen, starch, oligose, dextran, Mierocrystalline cellulose, agarose, chitosan, mucopolysaccharide, peptidoglycan.
The hybrid reaction temperature of described reagent and biological specimen is about 50 ℃～80 ℃.
The mixing acid base number of described biological specimen and reagent is pH 6～9.
Described biological specimen includes one or more nucleic acid.
Described biological specimen includes cellular material.
Described biological specimen comprises prokaryotic organism, fungi, virus, cell, blood, amniotic fluid, cerebrospinal fluid, or from the tissue and the tissue juice thereof of skin, muscle, conjunctiva, placenta or intestines and stomach.
Method of the present invention is carried out a wash procedure after also can being included in described biological specimen and reagent mix.After this method also is included in described wash procedure, carries out one and dash the program of putting forward.
Cover group of the present invention can comprise that also a cleaning buffer solution and/or is towards carrying damping fluid.Wherein said cleaning buffer solution can be removed salt and protein.Wherein said dash carried damping fluid and nucleic acid can be washed by the solid phase carrier upper punch.
In the cover group of the present invention, described container is used for the splendid attire separation agent.
For above and other objects of the present invention, feature and advantage can be become apparent, hereinafter the spy enumerates preferred embodiment, and conjunction with figs., elaborates.
Description of drawings
Fig. 1 is the electrophoretic analysis of DNA agar-agar.
Embodiment
The present invention provides the method for a kind of separation and/or purification of nucleic acid.In the method for the invention, a separation agent is mixed with a biological sample and react the separable nucleic acid that goes out in the biological specimen in back.
In one embodiment, separation agent of the present invention comprises the polyoxyethylene glycol (PEG) of 1～40wt%, is preferably the polyoxyethylene glycol of 5～20wt%.
In another embodiment, separation agent of the present invention comprises the lower molecular weight alcohols greater than 30wt%, is preferably the lower molecular weight alcohols of 40～60wt%, and wherein the molecular weight of lower molecular weight alcohols is about 30～100g/mol.In one embodiment, the lower molecular weight alcohols is an ethanol.
In another embodiment, separation agent of the present invention can comprise polyoxyethylene glycol and lower molecular weight alcohols simultaneously.
Separation agent of the present invention more comprises a salt, a tensio-active agent, a proteolytic enzyme and a solid phase carrier.
Salt of the present invention can be any salt that is applicable to the molecular biosciences experiment, comprises an alkali metal group or alkaline earths and/or amine salt, for example, and Li
+, Na
+, K
+, Rb
+, Cs
+, Fr
+, Gu
+(guanidine), Be
2+, Mg
2+, Ca
2+, Sr
2+, Ba
2+, Ra
2+And/or NH
4 +In one embodiment, salt of the present invention can comprise a haloid element, is in particular Cl
-In general, Chang Yong salt is Gu-HCl (guanidinehydrochloride), LiCl, NaCl, Tris-HCl, Gu-SCN (guanidine thiocyanate) or its analogue.
Tensio-active agent of the present invention can be ionogenic surfactant, amphoterics or nonionic surface active agent, wherein be preferably nonionic surface active agent and amphoterics, there is no particular restriction for the kind of nonionic surface active agent, be preferably the nonionic surface active agent of polyoxyethylene class (polyoxyethylene), as Tween-20 (polysorbas20), Triton X-100 (Triton X-100), NP40 (polyoxyethylene nonylphenol ether) or Brij35 (polyoxyethylene laurel ether) etc.The usage quantity or the concentration of nonionogenic tenside do not limit, for example, can be 0.01% to 40%, be preferably 5% to 20%, there is no particular restriction for the kind of amphoterics, is preferably CHAPS (3-{ (3-cholamidopropyl) dimethylammonio}-propanesulfonate, 3-[3-(courage amido propyl) dimethylamino] the propanesulfonic acid inner salt), the usage quantity or the concentration of amphoterics do not limit, and for example can be 0.01% to 20%, are preferably 1% to 10%.
Proteolytic enzyme of the present invention, can be any material with protein hydrolysate effect, for example thermolysin, stomach en-, trypsinase, bromeline (bromelain), alkaline hydrolysis proteolytic enzyme (Alcalase), Proteinase K (Protease K), flavor protease (Flavorzyme) or the beneficial auspicious proteolytic enzyme of alkalescence (Esperase) etc. are preferably Proteinase K (Protease K).
Separation agent of the present invention can add polyose (polysaccharides), comprise glycogen (glycogen), starch (starch), oligose (oligosaccharides), dextran (dextran), Mierocrystalline cellulose (cellulose), agarose (agarose), chitosan (chitosan), mucopolysaccharide (mucopolysaccharides) or peptidoglycan (peptidoglycan), the binding ability of small molecules nucleic acid and solid phase carrier be can increase, nucleic acid productive rate and purity improved.
Biological specimen of the present invention is a kind of biological specimen that contains nucleic acid.There is no particular restriction for biological specimen of the present invention, can be any sample with nucleic acid, karyomit(e) and/or plastid.In one embodiment, biological specimen of the present invention can be fungi, prokaryotic organism (bacterium), virus, zooblast or vegetable cell.In another embodiment, biological specimen of the present invention can be the blood, amniotic fluid, cerebrospinal fluid of animal or from the tissue juice of skin, muscle, oral mucosa, placenta, intestines and stomach or other organ.It should be noted that biological specimen of the present invention does not need via any pre-treatment, can be directly and the separation agent reaction, can isolate needed nucleic acid.
Nucleic acid of the present invention can be the DNA or the RNA molecule of any natural or synthetic, comprises single strand dna, double chain DNA molecule, RNA, LNA, PNA, nucleic acid-protein complex and/or nucleic acid-carbohydrate complex body etc.
Separation agent of the present invention is with after biological specimen mixes, and carries out a reactions steps under a suitable condition.The temperature of reactions steps can be room temperature, 50 ℃～80 ℃, is preferably 60～70 ℃; The pH value can be 6～9, is preferably 7～8, and the reaction times can be 5～30 minutes, is preferably 10～20 minutes, and the best is 15 minutes.It should be noted that biomaterial can directly react with separation agent of the present invention, and must not carry out any pre-treatment.
After adding separation agent, if biological specimen is a complex biological sample or a whole blood corpse or other object for laboratory examination and chemical testing that contains the cell kenel, separation agent can make cell rupture disengage nucleic acid, and the nucleic acid in the sample can be separated because solubleness changes, and does not need to carry out in advance that sample is centrifugal, the molten pre-treatment step such as split of cellular segregation, cell.Those skilled in the art can select for use any suitable method to be further purified nucleic acid, and for example: isolating nucleic acid can utilize alcohol wash to obtain more highly purified nucleic acid.
In another embodiment, isolating nucleic acid can be with after solid phase carrier combines, and dashes to put forward again.Separation agent of the present invention also can change the structure of nucleic acid except meeting changes the solubleness of nucleic acid, make nucleic acid be easier to paste on solid phase carrier, to reach isolating purpose.
Solid phase carrier of the present invention can be a kind of magnetic substance, metal, metal oxide, silicide, polymkeric substance.The surface of solid phase carrier can be modified by chemical bond.In a preferred embodiment, the surface of solid phase carrier has carbohydrate (hydrocarbon polymer), for example, and Mierocrystalline cellulose, soluble cotton, dextran or its analogue.In another embodiment, the surface of solid phase carrier has a hydrophobic grouping, for example, and C
1～C
30Alkyl or C
5～C
30Aryl etc.In another embodiment, the surface of solid phase carrier can have a hydrophilic radical, for example, and hydroxyl, carbonyl, carboxyl, ester class, amido, sulfenyl etc.
In addition, after reactions steps, can carry out a wash procedure in regular turn and dash the program of putting forward.
Wash procedure comprises adding one cleaning buffer solution, to remove unnecessary salt and protein.Cleaning buffer solution of the present invention can be any suitable cleaning buffer solution, and it is mainly protein, glycan class, lipid or the cell debris that removes on the nucleic acid.In general, cleaning buffer solution can comprise alcohols and salt, and alcohols can be ethanol or polyoxyethylene glycol.Salt can be sodium-chlor or its analogue that is lower than 3M.In general, the volume of cleaning buffer solution and wash number are good to be enough to removing impurity (protein, glycan class, lipid or cell debris) but do not influence nucleic acid output.
Behind wash procedure, can carry out one and dash the program of putting forward.Dash the program of putting forward and comprise that adding one is put forward nucleic acid towards carrying damping fluid by the solid phase carrier upper punch.Can be water, TE, TAE or TBE or its analogue towards carrying damping fluid.
The method of separation of the present invention and/or purification of nucleic acid is used single agents, finishes with one step, can obtain the nucleic acid of high purity and high yield fast, easily.The more important thing is that biological specimen does not need via any pre-treatment, can be directly and the separation agent reaction, can isolate needed nucleic acid.
The present invention provides a kind of separation agent of nucleic acid in addition, comprises the polyoxyethylene glycol (PEG) of 1～40wt% at least and/or greater than the lower molecular weight alcohols of 30wt%.
Separation agent of the present invention can further comprise above-mentioned salt, tensio-active agent, solid phase carrier and/or proteolytic enzyme.
The present invention also provides a kind of cover group of isolating nucleic acid, comprise a separation agent, it contains the polyoxyethylene glycol (PEG) of 1～40wt% and/or greater than lower molecular weight alcohols, salt, tensio-active agent and solid phase carrier, a cleaning buffer solution, the container of 30wt%, and working instructions.
[embodiment]
1. the separate nucleic acid of whole blood
The PEG (0%, 5%, 10%, 15%, 20%, 30%), salt that the separation agent that uses in this embodiment contains different concns is 3M guanidinesalt hydrochlorate (Gu-HCl), the tensio-active agent magnetic solid phase carrier as 5%Tween-20 and 0.5 μ g, and contains proteolytic enzyme and 6M urea.
The separation agent of the present invention of getting 720 μ l directly mixes with the human whole blood of 200 μ l, under 56 ℃, react after 10 minutes, separate nucleic acid can be bonded on the magnetic solid phase carrier, after removing the aqueous solution, washed with cleaning buffer solution, add 200 μ l aseptic deionized waters at last and after 10 minutes the nucleic acid that magnetic solid phase carrier upper punch puts forward is done analysis towards dike.The cleaning buffer solution that this embodiment uses is 70%EtOH.
As shown in Table 1, separation method of the present invention does not need the red blood corpuscle in the pre-treatment whole blood sample, can be effectively directly with DNA by separating in the whole blood, can in the human whole blood of 200 μ l, isolate about 1～3 μ g DNA, purity (A
260/ A
280) be about 1.3～1.6.
Table one, the isolating effect of DNA
| PEG concentration (%) | DNA fractional dose (μ g) | DNA purity (A 260/A 280) |
| ?0 | 0.73 | 1.31 |
| ?5 | 1.68 | 1.55 |
| ?10 | 3.21 | 1.62 |
| ?15 | 2.85 | 1.46 |
| ?20 | 2.12 | 1.35 |
| ?30 | 1.03 | 1.26 |
In addition, in separation agent, use Different concentrations of alcohol (40%, 50% and 60%), salt as 3M guanidinesalt hydrochlorate (Gu-HCl), tensio-active agent as the magnetic bead of 5%Tween-20 and 0.5 μ g as solid phase carrier, and containing proteolytic enzyme and 6M urea, cleaning buffer solution is 70% EtOH.Can isolate about 2 μ g DNA, purity (A from the human whole blood of 200 μ l
260/ A
280) be about 1.3～1.6.
2. the separate nucleic acid of whole blood and blood plasma
The separation agent that uses in this embodiment contains 10% PEG, salt and is 10%Tween-20 as 6M guanidinesalt hydrochlorate, tensio-active agent, and the urea of magnetic solid phase carrier, proteolytic enzyme and the 6M of 0.5 μ g.
The separation agent of the present invention of getting 720 μ l directly mixes with the human whole blood of 200 μ l or the blood plasma after the centrifugation, under 56 ℃, react after 10～15 minutes, separate nucleic acid can be bonded on the magnetic solid phase carrier, after removing the aqueous solution, washed with cleaning buffer solution, add 200 μ l aseptic deionized waters at last and after 10 minutes the nucleic acid that magnetic solid phase carrier upper punch puts forward is done analysis towards dike.The cleaning buffer solution that this embodiment uses is 70% EtOH.
In the control group of embodiment, with the MegaZorb of commercially available Promega company
Reagent cover group represent traditional separation method as positive control group, promptly comprises dissolving (lysis), extraction, precipitation, cleaning, towards 5 steps such as putting forward.Institute's separated DNA is analyzed, and its result as shown in Table 2.The result shows the plasma sample of separation agent of the present invention in whole blood sample or the centrifugal pre-treatment of process, all can be purified into the about 5 μ g of DNA, purity (A by 200 μ l samples
260/ A
280) being about 1.89, separation agent of the present invention can be directly used in the complex samples purification of nucleic acid, does not need the pre-treatment of centrifugal sample.
Table two, the isolating effect of DNA
In another embodiment, use identical separation agent, and with the MegaZorb of commercially available Promega company
Reagent cover group is as positive control group.Each experiment repeats 3 times.
By table three result as can be known, separation agent of the present invention and method can be effectively directly with DNA by separating in the whole blood, and its separating effect and commercially available prod are suitable, the separable DNA of going out of 200 μ l whole blood samples measures about 5 μ g, this has higher purity with separation agent of the present invention institute separated DNA, and repetitive error is less, purity (A
260/ A
280) be about 1.9 ± 0.051, the sample of nucleic acid of purifying can be applicable to the polymeric enzyme reaction analysis (Polymerase Chain Reaction, PCR).
Table three, the isolating effect of DNA
In another embodiment, separation agent of the present invention can at room temperature react, can be effectively directly with DNA by separating in the whole blood, and the separable DNA of going out of 200 μ l whole blood samples measures about 4 μ g, purity (A
260/ A
280) be about 1.7.
3. the separate nucleic acid of cell
The separation agent that uses in this embodiment contains 10% PEG, salt and is 6M guanidinesalt hydrochlorate (Gu-HCl), the tensio-active agent magnetic solid phase carrier as 10%Tween-20 and 0.5 μ g, and contains proteolytic enzyme and 6M urea.
The separation agent of the present invention of getting 720 μ l directly mixes with the CT26 mice colorectal cancer cell strain of 200 μ l, and total cellular score is about 8 * 10
5Cells/ml under 56 ℃, reacts after 10 minutes, separate nucleic acid can be bonded on the magnetic solid phase carrier, behind the removal aqueous solution, be washed with cleaning buffer solution, the aseptic deionized water that adds 200 μ l at last, is analyzed by the nucleic acid that puts forward that dashes on the magnetic solid phase carrier after 10 minutes towards dike.The cleaning buffer solution that this embodiment uses is 70% EtOH.Its result as shown in Table 4.In this embodiment, with the MegaZorb of commercially available Promega company
Reagent cover group is as positive control group.
By the result as can be known, separation agent of the present invention and method can be effectively directly with DNA by separating in the cell, approximately can be by 10
5Cell purification goes out the DNA of about 7 μ g, purity (A
260/ A
280) be higher than 1.8.
Table four, the isolating effect of DNA
Directly carry out the electrophoretic analysis of DNA agar-agar with above-mentioned by the sample of nucleic acid that cell extracted, its result as shown in Figure 1.Reagent of the present invention and MegaZorb
All show high-molecular weight DNA bright band (swimming lane 1: by MegaZorb
The nucleic acid that extracts; Swimming lane 2～3: the nucleic acid that extracts by reagent of the present invention; Swimming lane 4:1Kb DNA marker).By the direct DNA in the isolated cell of electrophoresis result separation agent of the present invention as can be known and method, and its DNA isolation has high yield and high purity.
Though the present invention discloses as above with preferred embodiment, so it is not in order to qualification the present invention, any those of ordinary skill in the art, without departing from the spirit and scope of the present invention, when doing a little change and retouching.
Claims (50)
1. the method for an isolating nucleic acid, this method comprises:
After the biological specimen that one separation agent and is contained nucleic acid mixed, described separation agent can make nucleic acid separate and be bonded on the solid phase carrier in biological sample, it is characterized in that:
Described separation agent comprises the polyoxyethylene glycol of 1～40wt% and/or greater than lower molecular weight alcohols, a salt and the tensio-active agent of 30wt%, it makes nucleic acid be bonded to described solid phase carrier by the solubleness that changes nucleic acid.
2. the method for isolating nucleic acid as claimed in claim 1, the molecular weight of wherein said lower molecular weight alcohols is 30～100g/mol.
3. the method for isolating nucleic acid as claimed in claim 1, wherein said lower molecular weight alcohols is an ethanol.
4. the method for isolating nucleic acid as claimed in claim 1, the weight concentration of wherein said lower molecular weight alcohols is 40～60%.
5. the method for isolating nucleic acid as claimed in claim 1, the weight concentration of wherein said polyoxyethylene glycol is 5～20%.
6. the method for isolating nucleic acid as claimed in claim 1, wherein said solid phase carrier comprises metal, metal oxide, silicide or polymkeric substance.
7. the method for isolating nucleic acid as claimed in claim 1, wherein said solid phase carrier comprises magnetic substance.
8. the method for isolating nucleic acid as claimed in claim 1, wherein said salt comprises alkali metal group, alkaline earths and/or ammonium salt halogenide.
9. the method for isolating nucleic acid as claimed in claim 1, wherein said tensio-active agent comprises Tween-20, Triton X-100, NP40, Brij35 or CHAPS.
10. the method for isolating nucleic acid as claimed in claim 1, wherein said reagent also comprises a proteolytic enzyme.
11. the method for isolating nucleic acid as claimed in claim 1, wherein said reagent also comprises a carbohydrate.
12. the method for isolating nucleic acid as claimed in claim 11, wherein said reagent comprises a polyose or polysaccharide derivatives.
13. the method for isolating nucleic acid as claimed in claim 12, wherein said polyose comprises glycogen, starch, oligose, dextran, Mierocrystalline cellulose, agarose, chitosan, mucopolysaccharide, peptidoglycan.
14. the method for isolating nucleic acid as claimed in claim 1, the hybrid reaction temperature of wherein said reagent and biological specimen are 50 ℃～80 ℃.
15. the method for isolating nucleic acid as claimed in claim 1, the mixing acid base number of wherein said biological specimen and reagent is pH 6～9.
16. the method for isolating nucleic acid as claimed in claim 1, wherein said biological specimen includes one or more nucleic acid.
17. the method for isolating nucleic acid as claimed in claim 1, wherein said biological specimen includes cellular material.
18. the method for isolating nucleic acid as claimed in claim 1, wherein said biological specimen comprises prokaryotic organism, fungi, virus, cell, blood, amniotic fluid, cerebrospinal fluid, or from the tissue and the tissue juice thereof of skin, muscle, conjunctiva, placenta or intestines and stomach.
19. the method for isolating nucleic acid as claimed in claim 1 after this method also is included in described biological specimen and reagent mix, is carried out a wash procedure.
20. the method for isolating nucleic acid as claimed in claim 19 after this method also is included in described wash procedure, is carried out one and is dashed the program of putting forward.
21. the reagent of an isolating nucleic acid, this reagent comprise the polyoxyethylene glycol of 1～40wt% and/or greater than lower molecular weight alcohols, a salt, a tensio-active agent and the solid phase carrier of 30wt%.
22. the reagent of isolating nucleic acid as claimed in claim 21, the molecular weight of wherein said lower molecular weight alcohols are 30～100g/mol.
23. the reagent of isolating nucleic acid as claimed in claim 21, wherein said lower molecular weight alcohols is an ethanol.
24. the reagent of isolating nucleic acid as claimed in claim 21, the weight concentration of wherein said lower molecular weight alcohols are 40～60wt%.
25. the reagent of isolating nucleic acid as claimed in claim 21, the weight concentration of wherein said polyoxyethylene glycol are 5～20wt%.
26. the reagent of isolating nucleic acid as claimed in claim 21, wherein said salt comprise alkali metal group, alkaline earths and/or ammonium salt halogenide.
27. the reagent of isolating nucleic acid as claimed in claim 21, wherein said tensio-active agent comprise Tween-20, Triton X-100, NP40, Brij35 or CHAPS.
28. the reagent of isolating nucleic acid as claimed in claim 21, wherein said solid phase carrier comprises metal, metal oxide, silicide or polymkeric substance.
29. the method for isolating nucleic acid as claimed in claim 21, wherein said solid phase carrier comprises magnetic substance.
30. the reagent of isolating nucleic acid as claimed in claim 21, wherein this reagent also comprises a proteolytic enzyme.
31. the reagent of isolating nucleic acid as claimed in claim 21, wherein this reagent also comprises a carbohydrate.
32. the reagent of isolating nucleic acid as claimed in claim 31, wherein this reagent comprises a polyose or polysaccharide derivatives.
33. the reagent of isolating nucleic acid as claimed in claim 32, wherein said polyose comprises glycogen, starch, oligose, dextran, Mierocrystalline cellulose, agarose, chitosan, mucopolysaccharide or peptidoglycan.
34. the cover group of an isolating nucleic acid, this cover group comprises:
One reagent comprises the polyoxyethylene glycol of 1～40wt% and/or greater than lower molecular weight alcohols, a salt, a tensio-active agent, a solid phase carrier and/or the proteolytic enzyme of 30wt%, and
One container.
35. the cover group of isolating nucleic acid as claimed in claim 34, the molecular weight of wherein said lower molecular weight alcohols are 30～100g/mol.
36. the cover group of isolating nucleic acid as claimed in claim 34, wherein said lower molecular weight alcohols is an ethanol.
37. the cover group of isolating nucleic acid as claimed in claim 34, the weight concentration of wherein said lower molecular weight alcohols are 40～60wt%.
38. the cover group of isolating nucleic acid as claimed in claim 34, the weight concentration of wherein said polyoxyethylene glycol are 5～20wt%.
39. the cover group of isolating nucleic acid as claimed in claim 34, wherein said salt comprise alkali metal group, alkaline earths and/or ammonium salt halogenide.
40. the cover group of isolating nucleic acid as claimed in claim 34, wherein said tensio-active agent comprise Tween-20, Triton X-100, NP40, Brij35 or CHAPS.
41. the cover group of isolating nucleic acid as claimed in claim 34, wherein said solid phase carrier comprises metal, metal oxide, silicide or polymkeric substance.
42. the cover group of isolating nucleic acid as claimed in claim 34, wherein said solid phase carrier comprises magnetic substance.
43. the cover group of isolating nucleic acid as claimed in claim 34, wherein said reagent also comprises a proteolytic enzyme.
44. the cover group of isolating nucleic acid as claimed in claim 34, wherein said reagent also comprises a carbohydrate.
45. the cover group of isolating nucleic acid as claimed in claim 44, wherein said reagent comprises a polyose or polysaccharide derivatives.
46. the cover group of isolating nucleic acid as claimed in claim 45, wherein said polyose comprises glycogen, starch, oligose, dextran, Mierocrystalline cellulose, agarose, chitosan, mucopolysaccharide or peptidoglycan.
47. the cover group of isolating nucleic acid as claimed in claim 34 wherein should the cover group comprise also that a cleaning buffer solution and/or was towards carrying damping fluid.
48. the cover group of isolating nucleic acid as claimed in claim 47, wherein said cleaning buffer solution can be removed salt and protein.
49. the cover group of isolating nucleic acid as claimed in claim 47, wherein said dash carried damping fluid and nucleic acid can be washed by the solid phase carrier upper punch.
50. the cover group of isolating nucleic acid as claimed in claim 34, wherein said container is used for the splendid attire separation agent.
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|---|---|---|---|
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|---|---|---|---|
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| CN111534511A (en) * | 2020-06-03 | 2020-08-14 | 沈阳德宇生物科技有限公司 | Reaction liquid for nucleic acid purification and recovery, nucleic acid recovery kit and application thereof |
| CN112321870A (en) * | 2020-11-06 | 2021-02-05 | 浙江工商大学 | Microneedle patch and preparation method and application thereof |
| WO2022135420A1 (en) * | 2020-12-23 | 2022-06-30 | 四川安可瑞新材料技术有限公司 | Preparation method for magnetic microspheres for nucleic acid extraction, prepared product and use thereof |
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2009
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| CN107058290A (en) * | 2017-04-28 | 2017-08-18 | 北京金豪制药股份有限公司 | A kind of method for extracting nucleic acid and its extracts reagent |
| CN111534511A (en) * | 2020-06-03 | 2020-08-14 | 沈阳德宇生物科技有限公司 | Reaction liquid for nucleic acid purification and recovery, nucleic acid recovery kit and application thereof |
| CN112321870A (en) * | 2020-11-06 | 2021-02-05 | 浙江工商大学 | Microneedle patch and preparation method and application thereof |
| CN112321870B (en) * | 2020-11-06 | 2022-11-18 | 浙江工商大学 | Microneedle patch and preparation method and application thereof |
| WO2022135420A1 (en) * | 2020-12-23 | 2022-06-30 | 四川安可瑞新材料技术有限公司 | Preparation method for magnetic microspheres for nucleic acid extraction, prepared product and use thereof |
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