CN111206073B - Nucleic acid extraction kit adopting silica bead method, and use method and application thereof - Google Patents
Nucleic acid extraction kit adopting silica bead method, and use method and application thereof Download PDFInfo
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- CN111206073B CN111206073B CN202010217345.8A CN202010217345A CN111206073B CN 111206073 B CN111206073 B CN 111206073B CN 202010217345 A CN202010217345 A CN 202010217345A CN 111206073 B CN111206073 B CN 111206073B
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 239000011324 bead Substances 0.000 title claims abstract description 69
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 63
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 63
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 54
- 238000000605 extraction Methods 0.000 title claims abstract description 41
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 39
- 239000006166 lysate Substances 0.000 claims abstract description 93
- 239000000243 solution Substances 0.000 claims abstract description 61
- 239000007788 liquid Substances 0.000 claims abstract description 49
- 238000001179 sorption measurement Methods 0.000 claims abstract description 40
- 108010067770 Endopeptidase K Proteins 0.000 claims abstract description 33
- 239000003480 eluent Substances 0.000 claims abstract description 30
- 239000000463 material Substances 0.000 claims abstract description 21
- 239000000725 suspension Substances 0.000 claims abstract description 21
- 238000005336 cracking Methods 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims description 31
- OVBJJZOQPCKUOR-UHFFFAOYSA-L EDTA disodium salt dihydrate Chemical compound O.O.[Na+].[Na+].[O-]C(=O)C[NH+](CC([O-])=O)CC[NH+](CC([O-])=O)CC([O-])=O OVBJJZOQPCKUOR-UHFFFAOYSA-L 0.000 claims description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 22
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 17
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 11
- 239000011780 sodium chloride Substances 0.000 claims description 11
- 238000005119 centrifugation Methods 0.000 claims description 7
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- ZPIRTVJRHUMMOI-UHFFFAOYSA-N octoxybenzene Chemical compound CCCCCCCCOC1=CC=CC=C1 ZPIRTVJRHUMMOI-UHFFFAOYSA-N 0.000 claims description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims description 6
- 239000002563 ionic surfactant Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000003945 anionic surfactant Substances 0.000 claims description 4
- 238000000197 pyrolysis Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 239000005543 nano-size silicon particle Substances 0.000 claims 1
- 239000000741 silica gel Substances 0.000 claims 1
- 229910002027 silica gel Inorganic materials 0.000 claims 1
- 235000012239 silicon dioxide Nutrition 0.000 claims 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical group [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 13
- 238000007654 immersion Methods 0.000 abstract description 3
- 238000000354 decomposition reaction Methods 0.000 abstract description 2
- 230000003993 interaction Effects 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000012487 rinsing solution Substances 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 30
- 108020004414 DNA Proteins 0.000 description 24
- 238000003776 cleavage reaction Methods 0.000 description 10
- 230000009089 cytolysis Effects 0.000 description 10
- 230000007017 scission Effects 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 229910052710 silicon Inorganic materials 0.000 description 9
- 239000010703 silicon Substances 0.000 description 9
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 8
- 239000007983 Tris buffer Substances 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 230000002349 favourable effect Effects 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 108091092878 Microsatellite Proteins 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003398 denaturant Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- -1 iron ions Chemical class 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- QZKRHPLGUJDVAR-UHFFFAOYSA-K EDTA trisodium salt Chemical compound [Na+].[Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O QZKRHPLGUJDVAR-UHFFFAOYSA-K 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002390 cell membrane structure Anatomy 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- CRWJEUDFKNYSBX-UHFFFAOYSA-N sodium;hypobromite Chemical compound [Na+].Br[O-] CRWJEUDFKNYSBX-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention provides a nucleic acid extraction kit by a silica bead method, and a preparation method and application thereof. The silica bead method nucleic acid extraction kit comprises a lysate A, a lysate B, a proteinase K solution, an adsorption bead suspension, a rinsing solution and an eluent; wherein the lysate A, the lysate B and the proteinase K solution are mixed to form a lysate. After the lysate A, the lysate B and the proteinase K solution are prepared in proportion, on one hand, the use volume of the lysate can be increased, and the complete immersion of the detection material is facilitated, so that the nucleic acid in the cells can be fully released; meanwhile, the problems of low cracking efficiency, visible light decomposition and the like caused by interaction between the cracking liquid A and the cracking liquid B can be solved, so that the cracking liquid fully and effectively cracks cells, the cracking efficiency is high, nucleic acid with higher concentration is obtained, the quality of the nucleic acid is improved, and the accuracy of subsequent operation is ensured.
Description
Technical Field
The invention relates to a nucleic acid detection technology, in particular to a nucleic acid extraction kit by a silica bead method, and a use method and application thereof.
Background
With the advancement of DNA testing technology, DNA extraction has become a key element affecting the success or failure of DNA testing. How to extract enough high-quality DNA from trace detection materials is always a great difficulty in the field of forensic DNA detection. The main methods of DNA extraction at present comprise a magnetic bead method based on an adsorption dissociation principle, a silica bead method, a column method based on molecular weight difference and a filter membrane method.
The principle of the silica bead method is similar to that of the magnetic bead method, and the process of extracting nucleic acid is basically the same and basically comprises four basic steps: (1) The strong protein denaturants such as guanidine thiocyanate are utilized to destroy cell membranes and nuclear membrane proteins of the detection material, release DNA and inactivate nuclease; (2) adding magnetic beads or silicon beads for specific adsorption; (3) Rinsing and purifying by controlling the pH value and the ionic strength of the washing liquid to remove the PCR inhibitor; (4) Changing the solution environment, dissociating DNA and magnetic beads or silicon beads in the dissolving solution, and re-dissolving the DNA, thereby achieving the purpose of extraction and purification. The successful application of the silica bead method to trace biological specimens such as exfoliated cells and difficult biological specimens such as bones and teeth is accepted by many students.
However, at present, the existing silica bead method nucleic acid extraction kit in the market is few and the quality is uneven, so that the market ratio of the magnetic bead method is larger than that of the silica bead method in general. Although the market is relatively small, the silica bead method has particular advantages in nucleic acid extraction: 1. the anti-inhibition capability is strong, and the anti-inhibition agent is applicable to complex legal medical expert detection materials such as highly spoilage pollution and the like; 2. can carry out bead amplification, and effectively improves the extraction sensitivity.
CN105462961a discloses a method and kit for extracting nucleic acid. Adding a lysate to a sample to be extracted for cracking to release nucleic acid molecules, and then adding a material for adsorbing nucleic acid and a binding solution to adsorb the nucleic acid molecules to obtain a nucleic acid-adsorbing material compound; washing the nucleic acid-adsorbing material complex, eluting with an aqueous solution containing a surfactant as an eluent, and collecting the eluted solution to obtain the target nucleic acid molecule. Aiming at the defects of the existing method for extracting nucleic acid, the invention adds the surfactant into the eluent in the eluting link, thereby improving the yield of the eluted nucleic acid, obtaining more nucleic acid molecules, simultaneously preserving the extracted nucleic acid for a long time without being adsorbed on the pipe wall or degraded by nuclease, and improving the preservation time of the nucleic acid molecules.
CN109694863a discloses a lysate, a washing liquid, a nucleic acid extraction kit and a nucleic acid extraction method for nucleic acid extraction. The nucleic acid extraction kit comprises a lysate, wherein the lysate comprises 20-200mmol/L NaCl, 10-50mmol/L Tris, 10-50mmol/L EDTA, 0.1-0.5% sodium dodecyl sulfate, 2-5% TritonX-100 and 0.5-1mol/L NaBrO 3 And 0.5-1mol/L guanidine isothiocyanate. The nucleic acid extraction kit can extract nucleic acid with higher concentration.
Therefore, the silicon bead method nucleic acid extraction kit with high cleavage efficiency and high extraction efficiency is provided, DNA in a legal medical expert material can be effectively extracted, and a foundation is provided for DNA detection.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a silica bead method nucleic acid extraction kit, and a use method and application thereof. The DNA extracted by the silica bead method nucleic acid extraction kit has higher purity, better detection precision, simple preparation method and easy preservation.
To achieve the purpose, the invention adopts the following technical scheme:
the invention provides a silica bead method nucleic acid extraction kit, which comprises the following components: lysate A, lysate B, proteinase K solution, adsorption bead suspension, rinse solution and eluent;
wherein the lysate A, the lysate B and the proteinase K solution are mixed to form a lysate.
According to the invention, the lysate provided by the invention comprises the lysate A, the lysate B and the proteinase K solution, and after the solutions are prepared in proportion, the use volume of the lysate can be increased, the complete immersion of the detection material is facilitated, the lysate is enabled to fully and effectively lyse cells, the lysis efficiency is high, so that nucleic acid in the cells can be fully released, nucleic acid with higher concentration is obtained, the quality of the nucleic acid is facilitated to be improved, and the accuracy of subsequent operation is ensured.
Meanwhile, the cracking solution A and the cracking solution B are respectively prepared, so that the problem of low cracking efficiency caused by interaction between the cracking solutions A and B can be solved, the problem of visible light decomposition generated in the storage process of the cracking solution B is also reduced, and the kit provided by the invention is easy to store.
The lysate A comprises any one or more than two of tris (hydroxymethyl) aminomethane, sodium chloride or disodium ethylenediamine tetraacetate dihydrate.
Wherein, the tris (hydroxymethyl) aminomethane is a common nucleic acid solvent, and can stabilize the pH of the solution; sodium chloride provides the necessary ionic environment to provide sodium ions for subsequent nucleic acid DNA to bind to the silica beads; disodium ethylenediamine tetraacetate is used as a metal chelating agent, and can chelate heavy metal magnesium ions, iron ions and the like possibly existing in the solution, so as to protect the integrity of DNA.
Preferably, the lysate A comprises tris (hydroxymethyl) aminomethane with a molar concentration of 0.005-0.015M, sodium chloride with a molar concentration of 0.05-0.15M and disodium edetate dihydrate with a molar concentration of 0.005-0.015M.
The molar concentration of the tris (hydroxymethyl) aminomethane in the lysate A is 0.005-0.015M, for example, 0.005M, 0.006M, 0.008M, 0.01M, 0.012M, 0.014M or 0.015M.
The molar concentration of sodium chloride in the lysate A is 0.05-0.15M, for example, 0.05M, 0.06M, 0.08M, 0.1M, 0.12M, 0.14M or 0.015M, etc.
Disodium edetate dihydrate (EDTANa) in the lysate A 2 ·2H 2 O) is 0.005 to 0.015M, for example, 0.005M, 0.006M, 0.008M, 0.01M, 0.012M, 0.014M, 0.015M, or the like.
As a preferred technical scheme of the invention, the lysate B comprises an ionic surfactant. Ionic surfactants can disrupt cell membrane structure, denature proteins, releasing nucleic acid DNA into solution.
Preferably, the ionic surfactant is an anionic surfactant.
Preferably, the anionic surfactant is Sodium Dodecyl Sulfate (SDS).
Preferably, the mass concentration of the sodium dodecyl sulfate in the lysate B is 8-12%, for example, 8%, 8.2%, 8.4%, 8.5%, 8.8%, 9%, 9.5%, 10%, 10.4%, 10.5%, 10.8%, 11%, 11.2%, 11.5%, 11.8% or 12%, etc.
Preferably, the concentration of proteinase K in the proteinase K solution is 8-12mg/mL, for example, 8mg/mL, 8.5mg/mL, 9mg/mL, 9.5mg/mL, 10mg/mL, 10.5mg/mL, 11mg/mL, 11.5mg/mL, 12mg/mL, etc.
Proteinase K is a biological agent with strong digestion protein, can digest protein combined with DNA, and can make DNA be released and dissolved in solution.
According to the preferred technical scheme of the invention, the lysate A, the lysate B and the proteinase K solution are mixed in a volume ratio of (4-6): 1 (0.4-0.6) to form a lysate, for example, 4:1:0.4, 4.2:1:0.4, 4.4:1:0.4, 4.6:1:0.4, 4.8:1:0.4, 5:1:0.4, 5.2:1:0.4, 5.4:1:0.4, 5.6:0.4, 5.8:1:0.4, 6:1:0.42, 4:1:0.44, 4:1:0.46, 4:1:0.48, 4:1:0.5, 4:1:0.52, 4:1:0.55, 4:1:0.56, 4:1:0.58, 4:1:0.6:1:0.5, 4:1:0.6:1:0.6:1:0.6, 6:1:0.6:1.6, etc.).
As a preferred embodiment of the present invention, the eluent comprises tris and/or disodium edetate dihydrate. The eluent was used to re-release the DNA from the silica beads into the eluent.
Preferably, the eluent comprises the trishydroxymethyl aminomethane with the molar concentration of 8-12mM and the disodium ethylenediamine tetraacetate dihydrate with the molar concentration of 0.08-0.12 mM.
In the eluent, the molar concentration of disodium ethylenediamine tetraacetate dihydrate is 0.08-0.12mM, and the concentration is favorable for releasing DNA attached on the silica beads into the eluent, so that the subsequent PCR treatment is facilitated.
The molar concentration of the tris in the eluent is 8-12mM, for example, 8mM, 8.2mM, 8.5mM, 9mM, 9.2mM, 9.5mM, 10mM, 10.2mM, 10.5mM, 10.8mM, 11mM, 11.5mM or 12 mM.
The molar concentration of disodium ethylenediamine tetraacetate dihydrate in the eluent is 0.08-0.12mM, and can be, for example, 0.08mM, 0.082mM, 0.085mM, 0.09mM, 0.092mM, 0.095mM, 0.1mM, 0.102mM, 0.105mM, 0.108mM, 0.11mM, 0.115mM or 0.12mM, etc.
Preferably, the adsorption beads are silica nanoparticles modified with silica hydroxyl groups.
Preferably, the particle size of the nano-silica particles is 150 to 250nm, and may be, for example, 150nm, 160nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 250nm, or the like.
The silica nanoparticle modified by the silicon hydroxyl group has smaller specific surface area, is favorable for adsorbing DNA, and can avoid the influence of other impurities in the biological detection material.
As a preferable technical scheme of the invention, the adsorption liquid comprises any one or more than two of guanidine isothiocyanate, tris (hydroxymethyl) aminomethane, disodium ethylenediamine tetraacetate dihydrate or polyethylene glycol octyl phenyl ether.
Preferably, the adsorption liquid comprises guanidine isothiocyanate with the molar concentration of 3-5M, tris (hydroxymethyl) aminomethane with the molar concentration of 0.04-0.06M, disodium ethylenediamine tetraacetate dihydrate with the molar concentration of 0.01-0.03M and polyethylene glycol octyl phenyl ether with the mass fraction of 2-3 wt%.
Wherein, the guanidine isothiocyanate is a powerful chaotropic agent and a denaturant, and is used for providing a high-salt environment for the solution and promoting the specific combination of DNA and silicon beads; polyethylene glycol octyl phenyl ether is a nonionic surfactant used for dissolving lipid and dissociating protein.
The molar concentration of guanidine isothiocyanate in the adsorption solution is 3-5M, and can be 3M, 3.2M, 3.4M, 3.5M, 3.6M, 3.8M, 4M, 4.2M, 4.5M, 4.8M or 5M, for example.
The molar concentration of the tris (hydroxymethyl) aminomethane in the adsorption liquid is 0.04-0.06M, and may be, for example, 0.04M, 0.042M, 0.044M, 0.045M, 0.046M, 0.048M, 0.05M, 0.052M, 0.054M, 0.056M, 0.058M, or 0.06M.
The molar concentration of disodium ethylenediamine tetraacetate dihydrate in the adsorption solution is 0.01-0.03M, and may be, for example, 0.01M, 0.012M, 0.014M, 0.015M, 0.016M, 0.018M, 0.02M, 0.022M, 0.024M, 0.026M, 0.028M, or 0.03M.
The mass fraction of the polyethylene glycol octyl phenyl ether in the adsorption liquid is 2-3wt%, and can be 2wt%, 2.1wt%, 2.2wt%, 2.4wt%, 2.5wt%, 2.6wt%, 2.8wt%, 3wt%, or the like.
As a preferable technical scheme of the invention, the rinsing liquid is an ethanol water solution with the mass fraction of 70wt%, and the rinsing liquid is used for removing salt ions and other impurities remained in the adsorption step.
In the present invention, the nucleic acid extraction kit may comprise the following solutions: 50-100mL of lysate A, 20-50mL of lysate B, 1-5mL of proteinase K solution, adsorption bead suspension, rinsing solution and eluent.
In a second aspect, the present invention provides a method for using the silica bead nucleic acid extraction kit according to the first aspect, the method comprising the steps of:
(1) Mixing a lysate A, a lysate B and a proteinase K solution to obtain a lysate, mixing the lysate with a material to be detected, and performing pyrolysis;
(2) Centrifuging the solution obtained in the step (1), adding the adsorption liquid, uniformly mixing, adding the adsorption bead suspension, and vibrating and uniformly mixing to obtain a suspension;
(3) Centrifuging the suspension, removing supernatant liquid, adding a rinsing liquid into the precipitate, uniformly mixing, centrifuging, and removing the rinsing liquid;
(4) Adding the eluent, and uniformly mixing to obtain an eluent containing the nucleic acid of the material to be detected.
Preferably, the volume of the lysate in step (1) is 350-450. Mu.L, for example, 350. Mu.L, 360. Mu.L, 380. Mu.L, 390. Mu.L, 400. Mu.L, 420. Mu.L, 440. Mu.L, 450. Mu.L, etc.
Preferably, the temperature of the cleavage in step (1) is 55-58 ℃, e.g. 55 ℃, 55.5 ℃, 55.8 ℃,56 ℃, 56.5 ℃, 57 ℃, 57.5 ℃ or 58 ℃ etc., preferably 56 ℃; the time is 20-30min, such as 20min, 22min, 24min, 25min, 26min, 28min or 30 min.
Preferably, the rotational speed of the centrifugation in the step (2) is 2500 to 3500g, for example, 2500g, 2600g, 2700g, 2800g, 2900g, 3000g, 3100g, 3200g, 3300g, 3400g or 3500g, etc., preferably 2800 to 3200g.
Preferably, the centrifugation time in the step (2) is 6-8min, for example, 6min, 6.2min, 6.4min, 6.5min, 6.6min, 6.8min, 7min, 7.2min, 7.4min, 7.8min or 8min, etc.
Preferably, the volume ratio of the suspension of the adsorption beads to the adsorption solution in the step (2) is 1 (25-30), for example, 1:25, 1:25.5, 1:26, 1:27, 1:27.5, 1:28, 1:28.5, 1:29, 1:29.5 or 1:30, etc.
Preferably, the rotational speed of both centrifuges in step (3) is 2500 to 3500g, for example 2500g, 2600g, 2700g, 2800g, 2900g, 3000g, 3100g, 3200g, 3300g, 3400g or 3500g, etc., preferably 2800 to 3200g.
Preferably, the time of two centrifugation in the step (3) is 6-10min, for example, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min or 10min, etc.
Preferably, the volume ratio of the adsorption bead suspension to the rinse liquid is 1 (35-40), and may be, for example, 1:35, 1:35.5, 1:36, 1:37, 1:38, 1:38.5, 1:39, or 1:40, etc.
Preferably, the eluent in step (4) is used in an amount of 15 to 50. Mu.L, for example, 15. Mu.L, 18. Mu.L, 20. Mu.L, 25. Mu.L, 30. Mu.L, 35. Mu.L, 38. Mu.L, 40. Mu.L, 45. Mu.L, 50. Mu.L, etc.
As a preferable technical scheme of the invention, the using method comprises the following steps:
(1) Mixing the lysate A, the lysate B and 8-12mg/mL proteinase K to form a lysate, wherein the volume ratio of the lysate A to the lysate B to the proteinase K solution is (4-6) 1 (0.4-0.6), taking 350-400 mu L of lysate to mix with the material to be detected in the centrifugal sleeve, and carrying out pyrolysis for 20-30min at 55-58 ℃;
(2) Centrifuging 2500-3500g of the solution obtained in the step (1) for 6-8min, adding an adsorption liquid, uniformly mixing, and then adding an adsorption bead suspension, wherein the volume ratio of the adsorption bead suspension to the adsorption liquid is 1 (25-30), placing at 25-28 ℃ for 10-15min, and vibrating and uniformly mixing every 5min during the period;
(3) Centrifuging 2500-3500g of the solution obtained in the step (1) for 6-10min, removing supernatant liquid, adding a rinsing liquid into the precipitate, wherein the volume ratio of the adsorption bead suspension to the rinsing liquid is 1 (35-40), uniformly mixing, centrifuging 2500-3500g for 6-10min, removing the rinsing liquid, and drying for 5-10min until no liquid is reflected on the surfaces of the adsorption beads, and the whole is white wall powder;
(4) Adding 15-50 mu L of eluent, uniformly mixing, and preserving heat at 55-58 ℃ for 15-20min to obtain an eluent containing nucleic acid of a material to be detected.
In a third aspect, the present invention provides the use of a silica bead nucleic acid extraction kit according to the first aspect for extracting trace amounts of DNA.
The numerical ranges recited herein include not only the above-listed point values, but also any point values between the above-listed numerical ranges that are not listed, and are limited in space and for the sake of brevity, the present invention is not intended to be exhaustive of the specific point values that the stated ranges include.
Compared with the prior art, the invention has the beneficial effects that:
the nucleic acid extraction kit provided by the invention comprises the lysate A, the lysate B and the proteinase K solution, wherein after the solutions are prepared in proportion, the use volume of the lysate can be increased, the complete immersion of the detection material is facilitated, the lysate can fully and effectively lyse cells, and the lysis efficiency is high; in the eluent, the molar concentration of the disodium ethylenediamine tetraacetate dihydrate is favorable for releasing DNA attached on the silicon beads into the eluent, and the subsequent PCR treatment is facilitated. In the use method provided by the invention, the silicon beads are separated in a proper mode, so that the separation efficiency of the silicon beads is improved, the sensitivity of DNA extraction is higher, and the inhibition resistance is good.
Drawings
FIG. 1 is a schematic diagram of the flow chart of the silica bead method nucleic acid extraction kit provided by the invention.
Detailed Description
To facilitate understanding of the present invention, examples are set forth below. It will be apparent to those skilled in the art that the examples are merely for aiding in the understanding of the present invention and should not be construed as limiting the invention in any way, and that implementations are within the scope of the invention without departing from the spirit of the invention as set forth in the foregoing and following claims.
Example 1
The embodiment provides a silica bead method nucleic acid extraction kit, the kit includes:
example 2
The embodiment provides a silica bead method nucleic acid extraction kit, the kit includes:
example 3
The embodiment provides a silica bead method nucleic acid extraction kit, the kit includes:
example 4
This example provides a silica bead method nucleic acid extraction kit, the kit differs from example 1 in that: the molar concentration of the tris (hydroxymethyl) aminomethane in the lysate A is 0.001M;
example 5
This example provides a silica bead method nucleic acid extraction kit, the kit differs from example 1 in that: the molar concentration of the tris (hydroxymethyl) aminomethane in the lysate A is 0.03M;
example 6
This example provides a silica bead method nucleic acid extraction kit, the kit differs from example 1 in that: the molar concentration of disodium ethylenediamine tetraacetate dihydrate in the lysate a was 0.001M;
example 7
This example provides a silica bead method nucleic acid extraction kit, the kit differs from example 1 in that: the molar concentration of disodium ethylenediamine tetraacetate dihydrate in the lysate A is 0.03M;
example 8
The difference from example 1 is that: the molar concentration of disodium ethylenediamine tetraacetate dihydrate in the eluent of the kit is 1mM.
Example 9
The difference from example 1 is that: the molar concentration of disodium ethylenediamine tetraacetate dihydrate in the eluent of the kit is 0.05mM.
Comparative example 1
The difference from example 1 is that: the kit comprises a lysis solution, a reagent kit and a reagent kit, wherein the lysis solution comprises 0.01M of tris (hydroxymethyl) aminomethane, 0.1M of sodium chloride, 0.01M of disodium ethylenediamine tetraacetate dihydrate, sodium dodecyl sulfate with the final concentration of 10wt% and proteinase K with the final concentration of 10 mg/mL; i.e. the lysate is not separated into lysate A, B and proteinase K for storage.
Comparative example 2
The difference from example 1 is that: the kit comprises a lysis solution and a proteinase K solution of 10 mg/mL;
the lysate contained 0.01M tris, 0.1M sodium chloride, 0.01M disodium edetate dihydrate, sodium dodecyl sulfate at a final concentration of 10 wt%.
Comparative example 3
The difference from example 1 is that: the kit comprises a lysis solution A and a lysis solution B;
the lysate A comprises 0.01M of tris, 0.1M of sodium chloride and 0.01M of disodium ethylenediamine tetraacetate dihydrate;
the lysate B contains sodium dodecyl sulfate with a final concentration of 10wt%, and proteinase K with a concentration of 10 mg/mL.
Comparative example 4
The difference from example 1 is that: the kit comprises a lysis solution A and a lysis solution B;
the lysate A comprises 0.01M tris, 0.1M sodium chloride, 0.01M disodium ethylenediamine tetraacetate dihydrate and 10mg/mL proteinase K
The lysate B contained sodium dodecyl sulfate at a final concentration of 10 wt%.
Comparative example 5
The difference from example 1 is that: the kit comprises a lysate A, a lysate B and 10mg/mL proteinase K.
The lysate A comprises 0.01M of tris (hydroxymethyl) aminomethane and 0.1M of sodium chloride;
the lysate B contains sodium dodecyl sulfate, disodium edetate dihydrate at a final concentration of 10 wt%.
Comparative example 6
The difference from example 1 is that: the kit replaces sodium dodecyl benzene sulfonate with sodium dodecyl sulfate.
Test example 1
This test example uses the detection kit provided in example 1 to extract DNA from a biological test material, and the DNA extraction procedure is as shown in fig. 1:
(1) Dripping 10 mu L of frozen venous blood sample diluted 1000 times onto a cotton swab head, putting the cotton swab head into a centrifugal sleeve, mixing a lysate A, a lysate B and 10mg/mL proteinase K according to the proportion of 5:1:0.5, dripping 400 mu L onto a detection material, covering a tube cover, and cracking for 20min at 56 ℃;
(2) Centrifuging at 3000g for 5min, opening the cover, adding 550 μl of adsorption liquid, covering the tube cover, mixing, adding 20 μl of fully suspended silica bead suspension, standing at 25deg.C for 15min, and shaking and mixing every 5min;
(3) Centrifuging at 3000g for 10min, removing supernatant liquid completely, adding 750 μl of rinse solution into the precipitate, covering with a tube cover, mixing completely, centrifuging at 3000g for 10min, and removing rinse solution as much as possible;
(4) Drying at 56 ℃ for 5min under the uncapping state until no liquid is reflected on the surface of the silicon beads, and the whole body is white wall powder;
(5) Adding 40 μl of eluent, covering with a tube cover, mixing, and maintaining at 56 deg.C for 15min; the eluted product can be directly beaded for downstream PCR amplification.
Test examples 2 to 9
The difference from test example 1 is that the nucleic acid extraction kits provided in examples 2 to 9 were used in the test examples, respectively.
Test example 10
The difference from test example 1 is that the mixing ratio of cleavage liquid A, cleavage liquid B and proteinase K at 10mg/mL in this test example was 6:1:0.6, and the other conditions and steps were the same as those in test example 1.
Test example 11
The difference from test example 1 is that the mixing ratio of cleavage liquid A, cleavage liquid B and proteinase K10 mg/mL in this test example was 4:1:0.4, and the other conditions and steps were the same as those in test example 1.
Test example 12
The difference from test example 1 is that the mixing ratio of cleavage liquid A, cleavage liquid B and proteinase K10 mg/mL in this test example is 1:1:1, and the other conditions and steps are the same as those in test example 1.
Test example 13
The difference from test example 1 is that the mixing ratio of cleavage liquid A, cleavage liquid B and proteinase K10 mg/mL in this test example was 8:1:1, and the other conditions and steps were the same as those in test example 1.
Comparative test examples 1 to 6
The difference from test example 1 is that the nucleic acid extraction kits provided in comparative examples 1 to 6 were used in the test examples, respectively.
Performance testing
The invention has the advantage of extracting efficiency through the STR locus number reaction kit. STR (short tandem repeat sequence, short tandem repeat) is a DNA polymorphism locus widely existing in human genome, and has been widely applied to the fields of forensic individual identification, parent identification and the like due to the characteristics of short gene fragment, high amplification efficiency, accurate judgment and the like.
Specific data for the number of STR sites (data obtained by analysis of ABI 3500 model capillary electrophoresis) are shown in table 1.
TABLE 1
As can be seen from the above table, the DNA obtained by extracting the nucleic acid extraction kit by the silica bead method has stable STR site number, and as compared with the embodiment 1 and the comparative examples 1-5, the invention divides the lysate in the kit into lysate A, lysate B and proteinase K solution, so that cells can be fully and effectively lysed, the lysis efficiency is high, nucleic acid in the cells can be fully released, and the improvement of the quality of the nucleic acid is facilitated; in addition, as is clear from the comparison of test example 1 and test examples 10 to 13, the ratio of the lysate A, the lysate B and the proteinase K solution is particularly important when preparing the lysate, and the ratio affects the extraction stability, and the effect is better when the ratio is close to 5:1:0.5.
The applicant states that the detailed process equipment and process flows of the present invention are described by the above examples, but the present invention is not limited to, i.e., does not mean that the present invention must be practiced in dependence upon, the above detailed process equipment and process flows. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. A silica bead nucleic acid extraction kit, characterized in that the silica bead nucleic acid extraction kit comprises:
lysate A, lysate B, proteinase K solution, adsorption bead suspension, rinse solution and eluent;
wherein, the lysate A, the lysate B and the proteinase K solution are mixed to form a lysate when in use;
the cracking liquid A consists of 0.005-0.015M of tris (hydroxymethyl) aminomethane, 0.05-0.15M of sodium chloride and 0.005-0.015M of disodium ethylenediamine tetraacetate dihydrate;
the lysate B is an ionic surfactant;
the ionic surfactant is an anionic surfactant;
the anionic surfactant is sodium dodecyl sulfonate;
the mass concentration of the sodium dodecyl sulfate in the lysate B is 8-12%;
the concentration of proteinase K in the proteinase K solution is 8-12mg/mL;
when the lysate A, the lysate B and the proteinase K solution are used, the volume ratio is (4-6): 1 (0.4-0.6) is mixed to prepare the lysate;
the eluent consists of 8-12mM of trimethylol aminomethane and 0.08-0.12mM of disodium ethylenediamine tetraacetate dihydrate;
the adsorption solution consists of guanidine isothiocyanate, tris (hydroxymethyl) aminomethane, disodium ethylenediamine tetraacetate dihydrate and polyethylene glycol octyl phenyl ether;
the rinsing liquid is an ethanol water solution with the mass fraction of 70 wt%.
2. The silica bead nucleic acid extraction kit of claim 1, wherein the adsorption beads are silica nanoparticle modified with silica hydroxyl groups;
the particle size of the nano silicon dioxide particles is 150-250nm.
3. The silica bead method nucleic acid extraction kit according to claim 2, wherein the adsorption solution consists of guanidine isothiocyanate with a molar concentration of 3-5M, tris (hydroxymethyl) aminomethane with a molar concentration of 0.04-0.06M, disodium edetate dihydrate with a molar concentration of 0.01-0.03M and polyethylene glycol octyl phenyl ether with a mass fraction of 2-3 wt%.
4. A method of using the silica bead nucleic acid extraction kit of any one of claims 1-3, comprising the steps of:
(1) Mixing a lysate A, a lysate B and a proteinase K solution to obtain a lysate, mixing the lysate with a material to be detected, and performing pyrolysis;
(2) Centrifuging the solution obtained in the step (1), adding the adsorption liquid, uniformly mixing, adding the adsorption bead suspension, and vibrating and uniformly mixing to obtain a suspension;
(3) Centrifuging the suspension, removing supernatant liquid, adding a rinsing liquid into the precipitate, uniformly mixing, centrifuging, and removing the rinsing liquid;
(4) Adding the eluent, and uniformly mixing to obtain an eluent containing the nucleic acid of the material to be detected.
5. The method of claim 4, wherein the volume of the lysate in step (1) is 350-450 μl;
the cracking temperature in the step (1) is 55-58 ℃ and the time is 20-30min;
the rotational speed of the centrifugation in the step (2) is 2500-3500g;
the centrifugation time in the step (2) is 6-8min;
the volume ratio of the adsorption bead suspension to the adsorption liquid in the step (2) is 1 (25-30).
6. The method of claim 5, wherein the centrifugation in step (2) is performed at a rotational speed of 2800g to 3200g.
7. The method of claim 6, wherein the rotational speed of both centrifuges in step (3) is 2500-3500g;
the time of two times of centrifugation in the step (3) is 6-10min;
the volume ratio of the adsorption bead suspension to the rinse liquid in the using method is 1 (35-40);
the eluent in the step (4) is used in an amount of 15-50 mu L.
8. The method of claim 7, wherein the rotational speed of both centrifuges in step (3) is 2800-3200g.
9. Use according to claim 6, characterized in that it comprises the following steps:
(1) Mixing the lysate A, the lysate B and 8-12mg/mL proteinase K to form a lysate, wherein the volume ratio of the lysate A to the lysate B to the proteinase K solution is (4-6) 1 (0.4-0.6), taking 350-400 mu L of lysate to mix with the material to be detected in the centrifugal sleeve, and carrying out pyrolysis for 20-30min at 55-58 ℃;
(2) Centrifuging 2500-3500g of the solution obtained in the step (1) for 6-8min, adding an adsorption liquid, uniformly mixing, and then adding an adsorption bead suspension, wherein the volume ratio of the adsorption bead suspension to the adsorption liquid is 1 (25-30), placing at 25-28 ℃ for 10-15min, and vibrating and uniformly mixing every 5min during the period;
(3) Centrifuging 2500-3500g of the solution obtained in the step (1) for 6-10min, removing supernatant liquid, adding a rinsing liquid into the precipitate, wherein the volume ratio of the adsorption bead suspension to the rinsing liquid is 1 (35-40), uniformly mixing, centrifuging 2500-3500g for 6-10min, removing the rinsing liquid, and drying for 5-10min until no liquid is reflected on the surfaces of the adsorption beads, and the whole is white wall powder;
(4) Adding 15-50 mu L of eluent, uniformly mixing, and preserving heat at 55-58 ℃ for 15-20min to obtain an eluent containing nucleic acid of a material to be detected.
10. Use of the silica gel bead nucleic acid extraction kit of any one of claims 1 to 3 for extracting trace amounts of DNA.
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