CN1304589C - Process for separating nucleic acid from biological particles by solid-phase carrier - Google Patents
Process for separating nucleic acid from biological particles by solid-phase carrier Download PDFInfo
- Publication number
- CN1304589C CN1304589C CN 01140445 CN01140445A CN1304589C CN 1304589 C CN1304589 C CN 1304589C CN 01140445 CN01140445 CN 01140445 CN 01140445 A CN01140445 A CN 01140445A CN 1304589 C CN1304589 C CN 1304589C
- Authority
- CN
- China
- Prior art keywords
- solid phase
- nucleic acid
- biomone
- add
- phase adsorption
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 35
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 35
- 239000007790 solid phase Substances 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 32
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 31
- 239000002245 particle Substances 0.000 title claims abstract description 10
- 210000004027 cell Anatomy 0.000 claims abstract description 30
- 238000001179 sorption measurement Methods 0.000 claims abstract description 28
- 239000004005 microsphere Substances 0.000 claims abstract description 16
- 239000000725 suspension Substances 0.000 claims abstract description 9
- 239000012472 biological sample Substances 0.000 claims abstract description 7
- 238000005336 cracking Methods 0.000 claims abstract description 6
- 239000012528 membrane Substances 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 22
- 238000000197 pyrolysis Methods 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 238000000926 separation method Methods 0.000 claims description 11
- 239000012149 elution buffer Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- -1 small molecules nucleic acid Chemical class 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000003805 vibration mixing Methods 0.000 claims description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 4
- SPSSULHKWOKEEL-UHFFFAOYSA-N 2,4,6-trinitrotoluene Chemical group CC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O SPSSULHKWOKEEL-UHFFFAOYSA-N 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 239000012148 binding buffer Substances 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 230000009089 cytolysis Effects 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims description 2
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- BEGBSFPALGFMJI-UHFFFAOYSA-N ethene;sodium Chemical group [Na].C=C BEGBSFPALGFMJI-UHFFFAOYSA-N 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 2
- 239000007853 buffer solution Substances 0.000 claims 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 claims 1
- WGYFACNYUJGZQO-UHFFFAOYSA-N aminomethanetriol Chemical compound NC(O)(O)O WGYFACNYUJGZQO-UHFFFAOYSA-N 0.000 claims 1
- 210000003850 cellular structure Anatomy 0.000 claims 1
- 238000001976 enzyme digestion Methods 0.000 claims 1
- 210000004369 blood Anatomy 0.000 abstract description 13
- 239000008280 blood Substances 0.000 abstract description 12
- 239000002609 medium Substances 0.000 abstract description 7
- 241000700605 Viruses Species 0.000 abstract description 2
- 210000002919 epithelial cell Anatomy 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 abstract description 2
- 239000003104 tissue culture media Substances 0.000 abstract description 2
- 210000002700 urine Anatomy 0.000 abstract description 2
- 229920002477 rna polymer Polymers 0.000 abstract 2
- 206010036790 Productive cough Diseases 0.000 abstract 1
- 239000000969 carrier Substances 0.000 abstract 1
- 238000010276 construction Methods 0.000 abstract 1
- 238000003795 desorption Methods 0.000 abstract 1
- 210000003802 sputum Anatomy 0.000 abstract 1
- 208000024794 sputum Diseases 0.000 abstract 1
- 238000007885 magnetic separation Methods 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 238000001962 electrophoresis Methods 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- BGPVFRJUHWVFKM-UHFFFAOYSA-N N1=C2C=CC=CC2=[N+]([O-])C1(CC1)CCC21N=C1C=CC=CC1=[N+]2[O-] Chemical compound N1=C2C=CC=CC2=[N+]([O-])C1(CC1)CCC21N=C1C=CC=CC1=[N+]2[O-] BGPVFRJUHWVFKM-UHFFFAOYSA-N 0.000 description 3
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 3
- 239000004141 Sodium laurylsulphate Substances 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108091092562 ribozyme Proteins 0.000 description 3
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002205 phenol-chloroform extraction Methods 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- GJYPUXPGBAIWQC-UHFFFAOYSA-N C(Cl)(Cl)Cl.C1(=CC=CC=C1)O.C1=CC=CC=C1 Chemical compound C(Cl)(Cl)Cl.C1(=CC=CC=C1)O.C1=CC=CC=C1 GJYPUXPGBAIWQC-UHFFFAOYSA-N 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method using solid phase carriers to separate nucleic acids out of biological particles, which belongs to the technical field of fast separating biological particles. The method foris characterized in that Nano magnetic microsphere suspension or fibrous membrane which is especially processed is used as the solid phase adsorption medium to separate cells in biological particles, cell cracking solution is used for cracking cell structures, biological particles of white cells, virus particles, epithelial cells, culture cells, etc., which contain ribonucleic acids are directly enriched out of biological samples, such as whole blood, blood plasma, sputum, urine, cells, tissue culture media, etc., in the forms of solid phase adsorption and desorption, and ribonucleic acids are obtained. The method which has the advantages of convenience and high speed can be used for preparing various samples different in scale and variety, and is easy to realize the construction of automatic and miniaturized devices.
Description
Technical field
A kind ofly utilize cell that solid phase carrier method of isolating nucleic acid from biomone belongs to sharp separation and cracking biomone to obtain the technical field of nucleic acid.
Background technology
Based on biochip, integrated sample preparation on the technology platform of microarray, " miniature organism chip lab " that biological respinse and result detect is the advanced form of biochip technology, at present to biochemical reaction and as a result the chip of proofing unit make up and obtained bigger progress, but the sample preparation apparatus that how to make up automatization on chip remains a difficult problem.This be because: from whole blood, blood plasma, saliva, urine, enrichment contains nucleic acid in the biological samples such as cell and tissue culture medium, biomones such as the white corpuscle of target molecules such as albumen, virus particle, epithelial cell, culturing cell, lysing cell under the cell pyrolysis liquid effect again, method by extraction precipitation could obtain target molecule, wherein will pass through isolation of biological particles, and lysing cell structure and separate targets molecule be totally three steps.Want to realize sample separation automatization and microminiaturized target, operation scheme must satisfy following the requirement simultaneously: the volume of (1) sample and agents useful for same must be as far as possible little, (2) avoid centrifugally operated as far as possible, (3) operation steps is the least possible, (4) operating process is avoided introducing as far as possible and is polluted, (5) separation method has certain general applicability, but existing method for making sample is difficult to reach these requirements, as traditional method of isolating nucleic acid from whole blood is the benzene phenol-chloroform method, use centrifugal separation and toxic reagent.Existing method needs to use centrifugally operated mostly owing to do not solve the cellular segregation problem well; What have in the nucleic acid elution process also need heat up, and has also restricted its full process automatization.Sample often needs pre-treatment when separating polymer DNA, thereby step is many, is difficult to realize microminiaturized.
Summary of the invention
The object of the present invention is to provide a kind of solid phase carrier sharp separation biomone from biological sample that utilizes, with cell pyrolysis liquid lysing cell structure, with the elution buffer isolating nucleic acid or utilize microballon-polymerase chain reaction (PCR) technology to carry out target gene amplification, amplified production is stored in the method for the extraction separation nucleic acid among the liquid phase.
The invention is characterized in that it contains following steps successively:
(1) but in biological sample, add the solid phase adsorption medium that physical adsorption contains the cell of nucleic acid, it is that a kind of particle size range of suspension is 5～10000nm and through the nano-magnetic microsphere of organic coating, leaves standstill after making their rotation mixings;
(2) isolate the above-mentioned solid phase adsorption medium, abandoning supernatant is washed with damping fluid;
(3) the adding cell pyrolysis liquid makes itself and solid phase adsorption medium rotate mixing and leaves standstill back binding buffer liquid and makes lysis;
(4) with elution buffer washing above-mentioned solid phase adsorption medium, obtain above-mentioned solid phase adsorption medium---the mixture of nucleic acid;
(5) recovery contains the elution buffer of nucleic acid and isolates nucleic acid.
The mixture that described (4) step obtains is the template of the target gene amplification usefulness in a kind of " microballon-polymerase chain reaction (PCR) " technology.
For small molecules nucleic acid (the sub-DNA of RNA or plasmid), in (3) step, can in the supernatant liquor that discards, add the solid phase adsorption medium again, remove to digest unwanted small molecules nucleic acid with dna cleavage enzyme or RNA enzyme as required after, carry out successively (4), (5) more set by step.
Described solid phase adsorption material is at room temperature to use 0.3mol/l NaOH liquid soaked overnight, and then uses 0.5mol/lBCl
32H
2It is the neutral glass fibre membrane that the O immersion was washed till pH value through deionized water in 8 hours.
The method of described separation solid phase adsorption medium and liquid is the magnetic field separation method.
Described cell pyrolysis liquid contains: 2.5～4mol/l NaI, 3～5mol/l urea, 30～50g/l Triton-100 (trotyl), 8～12mmol/l EDTA (sodium ethylene diamine tetracetate) (pH6.5), 15～30mmol/l TrisHCl (Tutofusin tris) is (pH6.5).
Described cell pyrolysis liquid consists of: 15～20g GuSCN (guanidinium isothiocyanate), the 100ml TE of pH6.0 (10mmol/lEDTA, 25mmol/l TrisHCl), 15～30gPEG8000 (polyoxyethylene glycol).
Described elution buffer be TE solution (10mmol/l TrisHCl, 1mmol/lEDTA).
Use proof: it has reached intended purposes.
Description of drawings
Human genome DNA's electrophoretogram of Fig. 1: embodiment 3.The agarose electrophoresis of the genomic dna that extracts with 15mg/ml magnetic ball suspension from 300 μ, 1 people's whole blood detects figure.The amount of magnetic ball suspension; 1 (label among Fig. 2, down together) and 2,5 μ l; 3 and 4,10 μ l; 5 and 6,20 μ l; 7 and 8,30 μ 1; 9 and 10,50 μ l; 11 and 12,70 μ l; 13,100 μ l; 14,120 μ l; 15,150 μ l; M:1 μ l λ phage DNA (the HindIII enzyme is cut) marker.
Fig. 2: the genomic dna that extracts with the magnetic ball from people's whole blood carries out the agarose electrophoresis detection figure of HLA-A allelotrope pcr amplification after product.O: negative control, P: positive control.
Fig. 3: with the schematic flow sheet of magnetic microsphere DNA isolation from whole blood.
Specific implementation method
Embodiment 1: the separation of polymer DNA.Being example: in the little centrifuge tube (eppendorf) of high-temperature sterilization, add the nano-magnetic microsphere suspension 40 μ l of 15mg/ml and the two strands of 0.4 μ g/ μ l is gone into-dna molecular amount mark (marker) (the HindIII enzyme is cut) 15 μ l with nano-magnetic microsphere adsorption of DNA segment, slight vibration 15s adds 4molL again on the vortex vibrator
-1NaI solution 80 μ l, behind the 15s that slightly vibrates, left standstill 3 minutes.Add absorbefacient Virahol 100 μ l again, the 5s that slightly vibrates, after left standstill 2 minutes.With magnetic separation rack the magnetic ball is fixed, abandoning supernatant is with the nanometer magnetic ball of 100 μ l, 70% washing with alcohol adsorption of DNA 2 times.TE solution (the 10molL that adds 50 μ l, pH=8.0
-1Tris.Cl, 1mmolL
-1EDTA).62 ℃ of constant temperature 12 minutes elutes DNA from the magnetic ball in water-bath.Again magnetic ball and TE solution separating, the TE solution that the obtains horizontal detected through gel electrophoresis separated DNA of agarose segment.All operation only needs 15min, and the dna segment rate of recovery is more than 95%.
Embodiment 2: with genomic (genomic) DNA of nano-magnetic microsphere separating Escherichia coli.
Sample is the E.Coli coli strain that does not contain plasmid of overnight incubation.Get the cell culture fluid 300 μ l that obtain, add 50 μ l nano-magnetic microsphere suspension again.Vibration mixes the back and adopts magnetic separation rack to separate magnetic ball, abandoning supernatant.Add 300 μ l cell pyrolysis liquids (3mol/l NaI, 4mol/l urea, 30g/l Triton-100 (trotyl), 10mmol/l EDTA (pH6.5), 25mmol/l TrisHCl (pH6.5)) (down together) to the magnetic ball, shake up, room temperature leaves standstill 5min.Add 300 μ l Virahols.Slight vibration l5s on the vortex vibrator, room temperature leaves standstill 5min.With magnetic separation rack the magnetic ball is fixed, abandon supernatant after, wash magnetic microsphere 2 times with 70% ethanol again.Add above-mentioned 100 μ lTE solution (pH=6.0) then, room temperature leaves standstill 10min.With magnetic separation rack the magnetic ball is fixed again, collected elutriant, carry out ultra-violet analysis and agarose gel electrophoresis.DNA output is about 30 μ g/ml, adds the broken born of the same parents of Proteinase K with traditional sodium lauryl sulphate (SDS), and phenol one chloroform extraction method is compared, and output and purity are suitable.
Embodiment 3: with white corpuscle in the nano-magnetic microsphere fractionation by adsorption whole blood and extract wherein DNA.
This is tested used whole blood and is provided by healthy, with ACD (23mmol/l citric acid, 80mmol/l glucose, the 45mmol/l Trisodium Citrate) anti-freezing of blood 1/6 volume.Get an amount of blood 300 μ l, add the above-mentioned magnetic microsphere solution of 50 μ l (the TE solution with above-mentioned pH=6.0 suspends), slight vibration 15s after room temperature leaves standstill 3min, fixes the magnetic ball with magnetic separation rack on the vortex vibrator.Abandon other parts.Add 300 μ l cell pyrolysis liquids, the vibration mixing, room temperature leaves standstill 2min.Add 300 μ l Virahols vibration mixing again, room temperature leaves standstill 5min.With magnetic separation rack the magnetic ball is fixed, abandoned supernatant liquor.Behind 2 magnetic balls of 70% washing with alcohol, add the TE solution of above-mentioned pH6.0, with magnetic separation rack the magnetic ball is fixed behind the 10min, collect elutriant, carry out gel electrophoresis analysis, the ultraviolet spectrometry analysis.The genomic dna that extracts sepharose (0.9%) electrophoresis detection, electrophoretogram is seen accompanying drawing 1.This method has been set up a kind of method for preparing genomic dna from whole blood rapidly and efficiently, and it is without toxic reagent, operational safety.Elutriant that contains the human genome DNA that is used in the aforesaid operations respectively to be obtained or the magnetic bead that is adsorbed with genomic dna that is obtained directly carry out the allelic PCR test of known HLA-A (human leucocyte antigen) as template, and amplified production detects by agarose gel electrophoresis.Electrophoretogram is seen accompanying drawing 2.A kind of magnetic bead-PCR method (microsphere based PCR) of HLA amplified allele has rapidly and efficiently been set up in this test, compare more simply with traditional method for making sample, fast, the template preparation only needs 10min, and got rid of the PCR inhibitor, and do not had non-specific amplification.Fig. 3 is the schematic flow sheet of magnetic resolution method of the present invention.The 1st, white corpuscle, the 2nd, red corpuscle, the 3rd, magnetic ball, the 4th, DNA, the 5th, magnet.Magnetic ball 3 and the slight vibration of biological sample people whole blood are left standstill again, and magnetic ball 3 absorption white corpuscles 1 back is adsorbed on tube wall by magnet 5, discards all the other solution.Enter the process of cracking white corpuscle structure behind the adding cell pyrolysis liquid, behind the broken born of the same parents, after DNA4 is adsorbed by magnetic ball 3, under magnet 5 effects, be adsorbed on the tube wall, discard all the other solution, after with the TE elution buffer DNA being washed from the magnetic ball, the magnetic ball is fixing by magnet 5, and the TE solution that will contain DNA moves in the new pipe.To 300 μ l people whole bloods, adopt 100 μ l magnetic ball solution (15 μ g/ μ l) can obtain the result.
Embodiment 4: with the plasmid DNA of nano-magnetic microsphere separating Escherichia coli.
Sample is for cultivating the E.coli bacterial strain that contains plasmid that spends the night, testing sequence is: get the cell culture liquid 300 μ l that obtain, add 50 μ l nano-magnetic microsphere suspension (15 μ g/ μ l) then, vibration mixes the back and adopts magnetic separation rack to separate the magnetic ball, abandons solution, adds 300 μ l cell pyrolysis liquids to the magnetic ball, shake up, room temperature leaves standstill 5min, adds 300 μ l Virahols vibration mixing again, and room temperature leaves standstill 5min.With magnetic separation rack the magnetic ball is fixed, shifted supernatant liquor to another new pipe, add 50 μ l nano-magnetic microsphere suspension and 300 μ l Virahols again, shake up, room temperature leaves standstill 5min, with magnetic separation rack the magnetic ball is fixed, abandon supernatant, use 70% washing with alcohol magnetic ball 2 times again.The TE solution 100 μ l and an amount of RNA enzyme solution that add above-mentioned pH6.0 then, degradation of rna under optimal temperature.With magnetic separation rack the magnetic ball is fixed again, collected elutriant, carry out ultra-violet analysis and agarose electrophoresis and detect.Plasmid DNA output is about 5 μ g/ml, compares with traditional method, and output and purity are suitable.If with the RNA of nano-magnetic microsphere separating Escherichia coli, then available DNA enzyme replaces above-mentioned RNA enzyme, and all the other steps are identical.
Embodiment 5: with treated tunica fibrosa adsorption of DNA segment.
In the eppendorf of high-temperature sterilization pipe, add the glass fibre membrane after the processing be cut into 0.5 centimetre of circular film of diameter, on tunica fibrosa, add double-stranded λ-DNAmarker (the HandIII enzyme is cut) 15 μ l of 0.4mg/ μ l again, add 5molL again
-1NaI solution 80 μ l, the 15s that slightly vibrates leaves standstill 1min then.Add absorbefacient Virahol 100 μ l again, the 5s that slightly vibrates leaves standstill 2min.Remove supernatant liquor with centrifuging.With the diaphragm of 100 μ l, 70% washing with alcohol adsorption of DNA 2 times.Add 50 μ lTE solution (pH=8.0,10mmolL
-1Tris.Cl, 1mmol.L
-1EDTA).In water-soluble,, DNA is eluted from diaphragm with 62 ℃ of constant temperature 12min.Use centrifuging diaphragm and TE solution separating then, the TE solution that the obtains horizontal detected through gel electrophoresis separated DNA of agarose segment.Entire operation only needs 15min.
Embodiment 6: with the genomic dna of treated tunica fibrosa separating Escherichia coli.
Sample is the E.Coli bacterial strain that does not contain plasmid of overnight incubation.Get the cell culture fluid 300 μ l that obtain, add the fibre strip film diaphragm that is cut into wide 0.3 centimetre processing then.Draw abandoning supernatant again with suction pipe behind the vibration mixing.Add 300 μ l cell pyrolysis liquids (20gGuSCN, 100ml TE (pH6.0) (10mmolL
-1Tris.Cl, 25mmol.L
-1EDTA), 20gPEG8000), shake up, room temperature leaves standstill 5min.Add 300 μ l, 70% ethanol, slight vibration 15s on the vortex vibrator, room temperature leaves standstill 5min.Abandoning supernatant again after drawing with suction pipe is washed diaphragm 2 times with 70% ethanol again.Add the above-mentioned 100 μ l TE solution of pH=6.0 then, room temperature leaves standstill 10min.Collect elutriant, carry out ultra-violet analysis and agarose gel electrophoresis.DNA output is about 40 μ g/ml, adds the broken born of the same parents of Proteinase K with traditional SDS, and the phenol-chloroform extraction process is compared, and output and purity are suitable.
Claims (7)
1. method of utilizing solid phase carrier isolating nucleic acid from biomone contains and separates and the cellularstructure of cracking biomone and therefrom isolate the step of nucleic acid, it is characterized in that described method is made of successively following steps:
(1) but in biological sample, add the solid phase adsorption medium that physical adsorption contains the cell of nucleic acid, it is that a kind of particle size range of suspension is 5-10000nm and through the nano-magnetic microsphere of organic coating,
Leave standstill after making their rotation mixings;
(2) isolate the above-mentioned solid phase adsorption medium, abandoning supernatant is washed with damping fluid;
(3) add cell pyrolysis liquid, make it with above-mentioned solid phase adsorption medium rotation mixing and after leaving standstill, binding buffer liquid makes lysis;
(4) with lavation buffer solution washing above-mentioned solid phase adsorption medium, obtain a kind of by above-mentioned solid phase adsorption medium and nucleic acid bonded mixture;
(5) recovery contains the elution buffer of nucleic acid and isolates nucleic acid.
2. a kind of method of utilizing solid phase carrier isolating nucleic acid from biomone according to claim 1, it is characterized in that: for small molecules nucleic acid, in above-mentioned (3) step, in the above-mentioned supernatant liquor that discards, add the solid phase adsorption medium again, fall unwanted small molecules nucleic acid with dna cleavage or RNA enzymic digestion as required, carry out (4) (5) successively set by step again.
3. method of utilizing solid phase carrier isolating nucleic acid from biomone comprises successively and separates and the cell of cracking biomone and from from isolating the step of nucleic acid, it is characterized in that it contains following steps successively:
(1) but in biological sample, add the solid phase adsorption material that physical adsorption contains the cell of nucleic acid, described solid phase adsorption material is at room temperature to use 0.3mol/L NaOH liquid soaked overnight, and then uses 0.5mol/L BCl
32H
2The O immersion was washed till the pH value through deionized water in 8 hours and is the neutral glass fibre membrane, and their are slightly left standstill behind vibration mixing, and abandoning supernatant;
(2) add cell pyrolysis liquid, make it with above-mentioned solid phase adsorption medium rotation mixing and after leaving standstill, binding buffer liquid makes lysis;
(3) with lavation buffer solution washing above-mentioned solid phase sorbing material, obtain a kind of by above-mentioned solid phase sorbing material and nucleic acid bonded mixture;
(4) recovery contains the elution buffer of nucleic acid and isolates nucleic acid.
4. according to the method for utilizing solid phase carrier isolating nucleic acid from biomone of claim 1, it is characterized in that:
The method of described separation solid phase adsorption medium and liquid is the magnetic field separation method.
5. according to the method for utilizing solid phase carrier isolating nucleic acid from biomone of claim 1, it is characterized in that:
Described cell pyrolysis liquid contains: 2.5～4mol/L NaI, and 3～5mol/L urea, 30～50g/LTriton-100 are trotyl, 8～12mmol/L EDTA is a sodium ethylene diamine tetracetate, its pH is that 6.5,15～30mmol/L TrisHCl is the trihydroxy-aminomethane, and its pH is 6.5.
6. according to the method for utilizing solid phase carrier isolating nucleic acid from biomone of claim 1, it is characterized in that:
Described cell pyrolysis liquid consists of: 15～20g GuSCN is a guanidinium isothiocyanate, and pH is that 6.0 100ml TE is 10mmol/L EDTA and 25mmol/L TrisHCl, and 15～30g PEG8000 is a polyoxyethylene glycol.
7. according to the method for utilizing solid phase carrier isolating nucleic acid from biomone of claim 1, it is characterized in that:
Described elution buffer is that TE solution is 10mmol/L TrisHCl and 1mmol/LEDTA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01140445 CN1304589C (en) | 2001-12-07 | 2001-12-07 | Process for separating nucleic acid from biological particles by solid-phase carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01140445 CN1304589C (en) | 2001-12-07 | 2001-12-07 | Process for separating nucleic acid from biological particles by solid-phase carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1355319A CN1355319A (en) | 2002-06-26 |
| CN1304589C true CN1304589C (en) | 2007-03-14 |
Family
ID=4675871
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01140445 Expired - Fee Related CN1304589C (en) | 2001-12-07 | 2001-12-07 | Process for separating nucleic acid from biological particles by solid-phase carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1304589C (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1230531C (en) * | 2002-12-09 | 2005-12-07 | 清华大学 | Method and kit for separating cell particle from sample |
| EP1607748B1 (en) * | 2003-03-24 | 2013-05-15 | Sony Corporation | Nucleic acid extracting kit, and nucleic acid extracting method |
| EP1624306A4 (en) * | 2003-04-30 | 2008-12-24 | Chengdu Kuachang Medical Ind L | Apparatus including nanostructures used for separation or analysis, and the preparation and application thereof |
| CN100507522C (en) * | 2004-12-15 | 2009-07-01 | 中国科学院上海应用物理研究所 | Fluorescence detection method for DNA and kit thereof |
| CN101363045B (en) * | 2007-09-10 | 2012-10-17 | 厦门致善生物科技有限公司 | Method for separating and purifying biological target material using ultrafine magnet particle |
| CN101851617B (en) * | 2010-04-28 | 2012-11-21 | 长春市博坤生物科技有限公司 | Extracting method of viral nucleic acid capable of being used for high flux full automation extraction by magnetic bead method |
| CN105420818A (en) * | 2015-11-17 | 2016-03-23 | 安诺优达基因科技(北京)有限公司 | Fast construction method for next generation sequencing library based on magnetic bead binding |
| CN105441425B (en) * | 2015-12-18 | 2019-03-05 | 河北蓝梦生物医药科技有限公司 | A kind of extracting solution and method extracting RNA |
| EP3487869B1 (en) * | 2016-07-25 | 2023-09-06 | IST Innuscreen GmbH | Method for enriching biomolecules and for removing the biomolecules from a biological sample |
| CN112111391A (en) * | 2017-01-24 | 2020-12-22 | 北京万泰生物药业股份有限公司 | Micro-fluidic chip and convection PCR amplification detection system |
| CN107043767A (en) * | 2017-05-05 | 2017-08-15 | 谭淼 | A kind of method that nucleic acid is extracted based on nanometer multidimensional chromato-graphy solid phase carrier |
| WO2019100620A1 (en) * | 2017-11-24 | 2019-05-31 | 浙江今复康生物科技有限公司 | Method for storing sputum and method for rapidly extracting nucleic acid from sputum |
| CN109382051B (en) * | 2018-11-13 | 2020-12-18 | 中国化学赛鼎宁波工程有限公司 | Production system and production process of nano biomedical material |
| CN109351301B (en) * | 2018-11-13 | 2020-12-11 | 中国化学赛鼎宁波工程有限公司 | Production system and production method of nano biomedical material |
| CN111206030B (en) * | 2020-02-18 | 2022-08-16 | 南京农业大学 | Virus nucleic acid extraction method based on glass fiber filter paper |
| CN113584128A (en) * | 2021-07-29 | 2021-11-02 | 翌圣生物科技(上海)股份有限公司 | Solid phase particulate material for processing biological samples and biological sample processing method |
-
2001
- 2001-12-07 CN CN 01140445 patent/CN1304589C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1355319A (en) | 2002-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1304589C (en) | Process for separating nucleic acid from biological particles by solid-phase carrier | |
| US9624252B2 (en) | Selective nucleic acid fragment recovery | |
| EP2872523B1 (en) | Microorganism nucleic acid purification from host samples | |
| CN100564541C (en) | Preprocessed chip of porous silica microfluid sample | |
| CN102041255A (en) | Instrument-free nucleic acid extraction device and trace nucleic acid extraction method | |
| US11702648B2 (en) | Process for concentrating cells from a sample and then isolating nucleic acids from said cells | |
| CN201648403U (en) | Nucleic acid extraction apparatus without instruments | |
| Kendall et al. | Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption | |
| CN111154847A (en) | Rapid nucleic acid extraction sequencing identification method based on bacterial 16S rDNA sequence | |
| US8691559B2 (en) | Micro channel, device for recovering nucleic acid and method for recovering nucleic acid | |
| US20160376636A1 (en) | Compositions and methods for sample preparation | |
| CN1681925A (en) | Method for the enrichment of prokaryotic DNA | |
| CN101338312B (en) | Pellosil activate fluid and applications thereof for extracting nucleic acid | |
| CN116286791A (en) | Method for extracting virus DNA in soil | |
| US20240052336A1 (en) | Filter-based extracellular vesicle nucleic acid isolation method | |
| CN1314700C (en) | Process for extracting DNA from biological samples | |
| US20070073049A1 (en) | Method and apparatus for nucleic purification using ion-permeable polymer-coated electrode | |
| CN106916812A (en) | A kind of paramagnetic particle method Ago-Gel DNA extraction kit and its extracting method | |
| JP2007068534A (en) | Method for recovering nucleic acid, fine passage, and device for recovering nucleic acid | |
| WO2022114917A1 (en) | Method for isolating nucleic acid using binding buffer including compound having low or intermediate dielectric constant | |
| KR102629851B1 (en) | Polymer construct comprising space for biological material and method for purifying biological material using the same | |
| US7273705B2 (en) | Apparatus and method for separating ribonucleic acid | |
| JP2004514548A (en) | A multi-step method for collecting or removing biological samples by floating from an underlying colloidal medium | |
| CN103571938B (en) | The reverse transcription PCR method of tubercle bacillus affection is detected from clinical samples | |
| CN1291039C (en) | Process for extracting prokaryotic microorganism DNA in sample and special reagent thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070314 Termination date: 20191207 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |