CN110079523A - It is a kind of to extract the method for nucleic acid, Extraction medium, kit - Google Patents
It is a kind of to extract the method for nucleic acid, Extraction medium, kit Download PDFInfo
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- CN110079523A CN110079523A CN201910417756.9A CN201910417756A CN110079523A CN 110079523 A CN110079523 A CN 110079523A CN 201910417756 A CN201910417756 A CN 201910417756A CN 110079523 A CN110079523 A CN 110079523A
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Abstract
The present invention provides a kind of method for extracting nucleic acid, will be coupled for the antibody of target nucleic acid specificity with certain material, obtains nucleic acid extraction material；The nucleic acid extraction material is added in sample to be extracted, nucleic acid extraction material is specifically bound with nucleic acid, and then carries out nucleic acid extraction, is obtained nucleic acid-nucleic acid and is extracted material composite；It is washed with cleaning solution, the nucleic acid-nucleic acid for the impurity that is removed extracts material composite；It is eluted, obtains target nucleic acid molecules.The present invention also provides a kind of nucleic acid extraction medium, the nucleic acid extraction medium includes by the antibody for target nucleic acid and extracting the resulting nucleic acid extraction material of material coupling.The present invention also provides a kind of kits for extracting nucleic acid.The present invention defect low to the small fragment nucleic acid extraction rate of recovery for the prior art, utilize the characteristic of the capture of immune antiboidy specificity, specificity, it will be directed on the antibody coupling to certain material of target nucleic acid, then nucleic acid extracted and purified, improve the rate of recovery and efficiency of nucleic acid extraction.
Description
Technical field
The present invention relates to field of biotechnology, particularly relate to method, the Extraction medium, reagent of a kind of extraction nucleic acid
Box.
Background technique
1948, Mandel and Metais reported dissociative DNA in human blood (cell-free DNA, cfDNA) for the first time
Presence.CfDNA is the extracellular dna of a kind of cell-free state, fragmentation, is primarily present in blood, periosteum liquid and cerebrospinal fluid
In equal body fluid.Numerous studies discovery, cfDNA content increase to tumour getting up early diagnosis, noninvasive pre-natal diagnosis, examination of curative effect and prognosis
Judgement etc. has very high clinical value.In addition to this, cfDNA is in organ transplant, cerebral apoplexy, autoimmunity disease and cardiac muscle
Application value in the diseases such as infarct is also increasingly clear.Content of the cfDNA in blood plasma is extremely low, and approximate range is in 15-40ng/
ml。
There are Circulating tumor DNA (circulating tumor DNA) in tumor patient cfDNA.Have body thin on ctDNA
Cytoplasmic process change, DNA methylation, copy number variation etc. tumor-specific genes change information, thus ctDNA can be used as diagnosing tumor,
Detection, prognosis and the tool for instructing clinical individualization to be administered.
CfDNA content is few and height fragmentation, and most of cfDNA fragment length is in 166bp or so, wherein most source
In normal tissue, the only minute quantity ctDNA that is tumour source.The ctDNA in tumour source than normal tissue source cfDNA more
It is short.The cfDNA of < 150bp is more, wherein the ctDNA ratio contained is higher.Therefore, it obtains more multiple clips cfDNA and carries out downstream
Analysis can effectively improve the detection sensitivity of ctDNA.
The detection of cfDNA is divided into quantitative detection cfDNA concentration, gene mutation and detection cfDNA are complete in qualitative detection ctDNA
Whole sex index.In order to obtain reliable testing result, the cfDNA template of high quality is first had to, and cfDNA height fragmentation
Feature causes it to lose during the extraction process, and extraction difficulty is big, carries out the work such as medical diagnosis on disease to clinical application cfDNA and causes
It is difficult.Therefore, it is most important for clinical cfDNA detected downstream that concentration height, the more cfDNA of short-movie section are extracted.
Currently, many commercial kits can be used for cfDNA extraction, it is broadly divided into paramagnetic particle method and pellosil centrifugal column method,
It is all to rely on the non-specific binding between the silicone hydroxyl and cfDNA on earth silicon material surface and realize in principle
's.Most important product is the QIAamp free nucleic acid extracts kit (Qiagen CAN kit) of Qiagen company in the market.
The short nucleic acid fragment that it is 75bp and 91bp for length that correlative study, which shows, kit currently on the market extract
The higher rate of recovery is 31.8%-33.7%；The nucleic acid fragment for being 157bp for length, kit currently on the market extract
The rate of recovery is 40% or so；The middle longer nucleic acid segment for being 195bp and 303bp for length, kit currently on the market extract
The higher rate of recovery is 75.5%-77.6%；The nucleic acid long segment for being 398bp for length, kit currently on the market mention
Fetching yield is 76.0%-87.4%.
Although current nucleic acid extraction Technical comparing is mature, stringenter, such as unicellular genome is required for some
(the micro complete genome DNAs of isolated individual cells) or DNA amount itself are sequenced with regard to less, as fetus peripheral blood is swum
From DNA.Current method just has some limitations and deficiency for the nucleic acid extraction of these application fields.
Summary of the invention
For overcome the deficiencies in the prior art, it is an object of the present invention to provide a kind of method for extracting nucleic acid, using exempting from
Epidemic disease antibody specificity, specificity catching method extract nucleic acid, can be improved the extraction recovery to nucleic acid, especially for
The nucleic acid of small fragment high efficiency and can accurately capture corresponding nucleic, reach nucleic acid from complicated body fluid sample environment
The purpose of middle extraction and purifying.
A kind of method for extracting nucleic acid provided by the invention, includes the following steps:
Coupling will be coupled for the antibody of target nucleic acid with material is extracted, and obtain nucleic acid extraction material；
It extracts, sample to be extracted is added in the nucleic acid extraction material, the nucleic acid extraction material and sample to be extracted
Target nucleic acid specific binding in product, obtains nucleic acid-nucleic acid and extracts material composite；
Washing washs the nucleic acid-nucleic acid extraction material composite with cleaning solution, with the nucleic acid-core for the impurity that is removed
Acid extracts material composite；
The nucleic acid-nucleic acid after eluting, will be washed extracts material composite and elutes, to obtain target nucleic acid point
Son.
Preferably, the antibody for target nucleic acid includes anti-DNA antibody, anti-RNA antibody.
Preferably, the antibody for target nucleic acid include monoclonal antibody, it is polyclonal antibody, Antibody Fab fragment, single-stranded
Antibody, single domain antibody.
Wherein, the antibody for target nucleic acid further includes anti-DNA antibody analog, anti-RNA antibody analog；It is described
Antibody analog includes Antibody Fab fragment, single-chain antibody, single domain antibody, mimic peptide, aptamer.
Preferably, the nucleic acid includes methylate DNA, methylation RNA；The antibody for target nucleic acid includes anti-first
The antibody of the antibody of base DNA, anti-methylation RNA.
Preferably, the antibody of the anti-methylate DNA includes phonetic anti-5- methyl born of the same parents, anti-N6 methyl purine, anti-7- methyl
Guanine；The antibody of the anti-methylation RNA include anti-N6- methyl adenine, anti-1-methyladenosine, anti-7- methylguanosine,
Anti- 5- methylcytidine, anti-N2- dimethylguanosine, anti-5- (methylol) cytidine, anti-N- acetylcytosine nucleosides.
A second object of the present invention is to provide a kind of nucleic acid extraction medium, the nucleic acid extraction medium includes according to above-mentioned
Extract nucleic acid extraction material obtained by coupling step in nucleic acid.
Preferably, the nucleic acid extraction material can be directed to mesh with described to be natural or passing through chemical group modification
Mark the medium of the antibody coupling of nucleic acid；The chemical group include amino, carboxyl, phenyl, phenolic group, sulfydryl, hydroxyl, imidazole radicals,
Indyl, guanidine radicals.
Preferably, the material of the nucleic acid extraction material includes collagen, silk, cellulose, magnetic corpusculum, chitosan, dioxy
SiClx.
Preferably, the presentation structure of the nucleic acid extraction Material texture includes microporous barrier, microballoon, fiberfill, porous Jie
Matter, silicon substrate micro-fluidic chip.
Wherein, the microballoon includes magnetic bead, polystyrene microsphere, Sepharose microballoon, latex beads, polymethyl
Sour methyl esters microballoon.
Third object of the present invention is to provide a kind of kit for extracting nucleic acid, including present invention nucleic acid as described above
Extraction medium, cleaning solution, eluent.
Antibody of the present invention for target nucleic acid is to refer to and the target nucleic acid specificity knot in sample to be processed
The antibody of conjunction.
Nucleic acid extraction material of the present invention is the extraction material for referring to being coupled for target nucleic acid antibody,
The extraction Material texture includes but is not limited to natural or collagen, silk, cellulose, the magnetic of process chemical group modification are small
Body, chitosan, silica, the presentation structure for extracting Material texture include but is not limited to natural or by chemical groups
Microporous barrier, microballoon, magnetic bead, fiberfill, porous media, the silicon substrate micro-fluidic chip of modification.The present invention is by by the extraction
Material texture is prepared into microporous barrier, microballoon, magnetic bead, fiberfill, porous media and carries out occasionally with the antibody for target nucleic acid again
Connection, or the extraction Material texture is applied in the nucleic acid coating area in silicon substrate micro-fluidic chip again and for target nucleic acid
Antibody is coupled, so as to targetedly, efficiently extract the nucleic acid in sample to be processed.The wherein extraction material
It is widely used that magnetic bead, magnetic bead are a kind of super paramagnetic microspheres with fine particle size in the presentation structure of material, has superpower
Paramagnetism.
The present invention can be coupled described by using collagen, silk, cellulose, magnetic corpusculum, chitosan, silica etc.
Material for target nucleic acid antibody or the microporous barrier prepared to it, microballoon, magnetic bead, fiberfill, porous media carry out certain
Chemical group modification (such as amino, carboxyl, phenyl) after, then be coupled with for the antibody of target nucleic acid, be can be obtained
Nucleic acid extraction material of the invention, can be with nucleic acid interaction, and formation nucleic acid-nucleic acid that can be reversible extracts material composite,
And then it can be used for extracting nucleic acid.
Compared with prior art, the beneficial effects of the present invention are:
The antibody and extraction material are coupled for the antibody of target nucleic acid specific binding and are incorporated in one by selection
It rises, obtains nucleic acid extraction material, then nucleic acid extraction is carried out to sample to be extracted.It is specific, specificity using immune antiboidy
Catching method extracts nucleic acid, can capture corresponding nucleic with high efficiency and accurately, reaches nucleic acid from complicated body fluid sample
The purpose extracted and purified in this environment.Particularly with contain small fragment nucleic acid or the lower sample to be processed of nucleic acid content,
The extracting method of nucleic acid of the invention, Extraction medium, the kit for extracting nucleic acid have higher special identity, greatly
Improve rate of recovery when nucleic acid extraction.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, it is described in detail below with some embodiments.A specific embodiment of the invention
It is shown in detail by following embodiment.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes part of this application, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and do not constitute the improper restriction invented in fact this.In the accompanying drawings:
Fig. 1 is a kind of process flow chart of method for extracting nucleic acid of the invention；
Fig. 2 is the schematic illustration of the specific binding of the microballoon and nucleic acid of coupled antibody of the invention；
Fig. 3 is the schematic illustration of the specific binding of the centrifugal column film and nucleic acid of coupled antibody of the invention；
Fig. 4 is that fibroin protein film is coupled RNA specific polyclonal antibody centrifugal column method extraction cream in one embodiment of the invention
The process flow chart of the total serum IgE of adenocarcinoma cell；
Fig. 5 is that cyanogen bromide-activated agarose microbeads particle is coupled anti-DNA single-chain antibody extraction greatly in one embodiment of the invention
Amount extracts the process flow chart of the DNA of calf thymus；
Fig. 6 is the structural schematic diagram of silicon substrate micro-fluidic chip in one embodiment of the invention.
Fig. 7 is the agarose gel electrophoresis figure for the nucleic acid that the present invention extracts.
In figure: 1- microballoon；2- is directed to the antibody of target nucleic acid；3- nucleic acid；4- centrifugal column film；5- cyanogen bromide-activated agarose
The conjugate of microsphere particle and anti-DNA single-chain antibody；6- silicon base chip；7- well；8- fluid channel；The anti-methylate DNA of 9- is anti-
Body is coated with area；10- methylate DNA collection channel；11- heating module.
Specific embodiment
In order to which invention described herein can be more fully understood by, following embodiment is illustrated.It should understand that these embodiments
It being merely to illustrate property purpose and is understood not to limit the invention in any way.
Experimental raw, vessel used in the embodiment of the present invention can be purchased by routine business approach.
English name below herein is explained, table one is joined.
Table one
| Ultrapure water | Ultrapure water |
| TdT Buffer(5X) | Terminal enzyme (DNA) buffer |
| Biotin-11-dUTP | The triphosphoric acid deoxidation urine of biotin labeling is sweet |
| TdT | Terminal enzyme (DNA) |
| EDC | 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride |
| NHS | N-hydroxysuccinimide |
| MES | 2- (N- morpholino) ethanesulfonic acid |
| EDAC | Carbodiimide |
| PBS | Phosphate buffered saline solution |
| PBST | Phosphate buffer containing Tween-20 |
| EP pipe | Centrifuge tube |
| EDTA | Ethylenediamine tetra-acetic acid |
| Tris | Trishydroxymethylaminomethane |
| Triton X-100 | Triton X-100 |
| Tris-HCL | Trishydroxymethylaminomethane-hydrochloric acid |
| SDS | Lauryl sodium sulfate |
| NaHCO3 | Calcium bicarbonate |
| NaCl | Sodium chloride |
| NaOAc | Sodium acetate |
| Buffer LB | Sample-loading buffer |
| Buffer WB | Cleaning solution |
| Elution Buffer | Elution buffer |
A kind of method for extracting nucleic acid provided by the invention, as shown in Figure 1, including the following steps:
S1, coupling: it will be coupled for the antibody of target nucleic acid with material is extracted, obtain nucleic acid extraction material；
S2, extraction: sample to be extracted is added in the nucleic acid extraction material, the nucleic acid extraction material with it is to be extracted
Target nucleic acid specific binding in sample, obtains nucleic acid-nucleic acid and extracts material composite；
S3, washing: the nucleic acid-nucleic acid is washed with cleaning solution and extracts material composite, to be removed outside target nucleic acid
The nucleic acid-nucleic acid of impurity extracts material composite；
S4, elution: the nucleic acid-nucleic acid after will be washed extracts material composite and elutes, to obtain target core
Acid molecule.
As shown in Fig. 2, the schematic diagram of the specific binding for the microballoon and nucleic acid of coupled antibody, for the anti-of target nucleic acid
After body 2 and microballoon 1 are coupled, the microballoon of the coupled antibody of acquisition, using the specific binding of antibody and nucleic acid, thus extract to
Extract the nucleic acid 3 in sample.Wherein, the microballoon is natural or passing through chemical group modification, can be directed to described
The microballoon of the antibody coupling of target nucleic acid；The microballoon of the coupled antibody is a kind of nucleic acid extraction material of the invention.
As shown in figure 3, the schematic diagram of the specific binding for the centrifugal column film and nucleic acid of coupled antibody, for target nucleic acid
Antibody 2 and the coupling of centrifugal column film after, the centrifugal column film of the coupled antibody of acquisition, using the specific binding of antibody and nucleic acid,
To extract the nucleic acid 3 in sample to be extracted.Wherein, the centrifugal column film is natural or modifies by chemical group
, it can be with the centrifugal column film of the antibody coupling for target nucleic acid；The centrifugal column film of the coupled antibody is this hair
A kind of bright nucleic acid extraction material.
It should be appreciated that when the nucleic acid in sample to be processed in step S2 is not free state needing that lysate is added
It is cracked；When nucleic acid is free state, then nucleic acid can be directly extracted.
It should be appreciated that the elution step in step S3 can be with elution, it is also possible to high-temperature elution；Wherein
The temperature of high-temperature elution can control eluting temperature between 50-100 DEG C according to the length of target nucleic acid.
Using a kind of method for extracting nucleic acid of the invention, nucleic acid simply and is efficiently captured, by nucleic acid from sample to be processed
In extract, especially for the shorter nucleic acid of fragment length, still there is efficient extraction efficiency.
A kind of kit extracting nucleic acid provided by the invention, including present invention nucleic acid extraction medium as described above, washing
Liquid, eluent.
The cleaning solution for nucleic acid extraction that can be purchased on any existing market can be used in the cleaning solution, described to wash
What de- liquid can be used obtaining of can purchasing on any existing market can be by the target nucleic acid and for the antibody of target nucleic acid
Isolated eluent.
It should be appreciated that kit of the invention is also if the nucleic acids in samples to be processed of required extraction is not free state
Including lysate.
Extraction to nucleic acid is carried out using kit of the invention, the nucleic acid extraction medium in kit can specificity,
Specificity captures nucleic acid and is combined, so as to shorten nucleic acid extraction time, improve the rate of recovery of extracted nucleic acid.
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
The preparation of the polyclonal antibody of rabbit-anti double-stranded DNA
1. the preparation of Biotinylated double strand DNA
1) each reagent is sequentially added by two numeric order of table, the total volume of reagent is 50 μ L.
Table two
| Serial number | Reagent | Volume |
| 1 | Ultrapure water | 29μl |
| 2 | TdT Buffer(5X) | 10μl |
| 3 | DNA (1 μM) to be marked | 5μl |
| 4 | Biotin-11-dUTP | 5μl |
| 5 | TdT(10U/μl) | 1μl |
2) mixing is gently blown and beaten with rifle, is sure not whirlpool concussion, and 37 DEG C are incubated for 30 minutes.
3) 2.5 μ L probes are added and mark terminate liquid, mix gently termination reaction.
4) after probe label reaction terminating, 52.5 μ L chloroform-isoamyl alcohols (volume ratio 24:1) are added, whirlpool concussion makes to have
Machine phase and water phase are sufficiently mixed to extract TdT.
5) 12000-14000g is centrifuged 1-2 minutes, and it is spare to draw supernatant.Supernatant is by the DNA of biotin labeling.Gained
DNA length be 300bp.
2. the DNA of biotin labeling is reacted with Streptavidin
By 50 μ L biotinylated DNAs, the Streptavidin of the 1mg/mL of 100 μ L is added, 37 DEG C are incubated for 30 minutes, obtain
DNA- biotin-avidin complex antigen.
3. immune new zealand rabbit
DNA- biotin-avidin complex antigen is diluted to 100 μ g/mL, takes 500 μ L and isometric Freund's complete adjuvant
After mixing and being fully emulsified, disperses 15 points and subcutaneous inoculation injection is carried out to new zealand rabbit backbone two sides, 50 μ g's of per injection
Complex antigen.After initial immunity, at interval of two weeks booster immunizations, dosage as before, be immunized 4 times altogether.1st time immune Freund is complete
Adjuvant, other 3 immune incomplete Freund's adjuvants.Blood is taken to survey potency after the completion of immune, potency carries out again after reaching ideal value
Rabbit arteria carotis takes blood, and 5 000r/min are centrifuged 10min, separate serum, saves in -80 DEG C.Acquisition contains the more of rabbit-anti double-stranded DNA
The serum of clonal antibody.
4. the mostly anti-purifying of rabbit-anti double-stranded DNA
Serum 60mmol/L, the pH4.0 acetate buffer solution that need to be purified is taken to dilute 4 times；It is stirred at room temperature down in 30min
Octanoic acid is added dropwise, adds 75 μ l octanoic acids, 4 DEG C of standing 2h, 15 000r/min centrifugations by the serum volume before every milliliter of dilution
30min abandons precipitating；Add the pH 7.4,0.1mol/L PBS of 1/10 volume after the filtering of supernatant filter paper；In 30min under 4 DEG C of ice baths
Interior addition 0.277g/ml ammonium sulfate, makes into 45% saturation degree, and 4 DEG C stand overnight, 12 000r/min, centrifugation 30min, in abandoning
Clearly；Precipitating is dissolved in the PBS of appropriate 0.01mol/L, pH 7.4, is filtered with PD10 column, and efflux is collected, and be sterile filtered packing ,-
20 DEG C save backup, and the rabbit-anti double-stranded DNA obtained after purification is mostly anti-.
Embodiment 2
The preparation of the monoclonal antibody specific of anti-N6- adenylate methylation (m6A) RNA
1.m6A-RNA synthesis
5 ' the ends of 5uM 60base use existing conventional synthesis by the RNA of biotin modification by m6A modification, 3 ' ends
Method synthesis.
2. the N6- adenylate of biotin labeling methylates, (m6A) RNA is reacted with Streptavidin
N6- adenylate methylation (m6A) RNA of biotin labeling is taken 1 μM, 100 μ of Streptavidin of 1mg/mL is added
L, 37 DEG C are incubated for 30 minutes, and must methylate RNA- biotin-avidin complex antigen.
3. immune balb/c mice
It takes the 10 μ L of methylation RNA- biotin-avidin complex antigen after above-mentioned reaction to be diluted to 100 μ L with PBS to add
After the not formula adjuvant emulsion of equivalent, abdominal cavity multi-point injection is spaced second of injection in 4 weeks, third time is injected after 1 week.Finally with phase
The methylation RNA- biotin-avidin complex antigen of same dosage, through tail vein booster immunization.Spleen is taken after 3 days, prepares to melt
It closes.
4. the preparation of feeder cells
In the previous day of fusion, normal non-immune mouse 1 is taken, the neck that breaks is put to death, abdominal cut skin is fixed after disinfection,
The abdomen of exposure peeling.Then 5mL DMEM injection abdominal cavity is lured into poly- abdominal cavity cell, is sucked back with original syringe, is put into 50mL
Centrifuge tube, 1000rpm10min abandon supernatant, add full DMEM liquid, be dispensed into 96 orifice plates by every hole 0.1mL.Set incubator mistake
Night, next day use.
5. fusion
The spleen of sterile taking-up is put into plate on 200 mesh steel meshes, and 10mL DMEM is added, with injection core that splenocyte is light
Net gently was squeezed, both obtains splenocyte suspension, 1000rpm, 10min abandon supernatant.It is suspended and is precipitated with DMEM liquid.Cell count postposition
It is spare in ice bath.Press the measurement 1x10 of cell 2:18Splenocyte and 1 × 107Myeloma cell is mixed in 50mL centrifuge tube,
1000rpm, 10min abandon supernatant, flick tube wall, and sediment is made to mix such as paste；0.7mL 50%PEG is added in 37 DEG C of water-baths
Then plus 20mL DMEM, room temperature 1000rpm 10min rush is melted, and, abandons supernatant.The complete DMEM culture solution that 20mL contains HAT is added
20mL is distributed into 96 porocyte culture plates for having feeder cells by every 100 μ L of hole, sets in incubator and cultivate.It is used after 3 days
HAT selection culture solution (xanthine, aminopterin, thymidine intermixture) partly change the liquid once, continuous 2 weeks；Change
With HT adaptability culture solution (intermixture of secondary sulphur purine and thymidine), when hybridoma clone it is long to 1/4 visual field when, into
The screening of row positive colony.Using finite gradient dilution method, hybridoma microcolony is diluted to 0.5, every hole cell.Continue to use
HAT Selective agar medium culture 5-7 days.It is agglomerating (there are about 20-30 cells) to its growth, take cell conditioned medium to screen positive hybridoma
Cell line.
The screening of the mouse monoclonal antibody of 6.m6A-RNA specificity
The m6A-RNA of synthesis is added in 96 orifice plates of flat nucleic acid height absorption, every hole with the concentration of 100ng/mL
50 μ L, 4 DEG C are coated with overnight.50 μ L addition is taken to be coated in 96 orifice plates of m6A-RNA the supernatant of hybridoma, 37 DEG C of reaction 1h,
Every hole is cleaned with 300 μ L PBST, and totally 3 times.The sheep anti mouse of HRP label, every 50 μ L of hole, 37 DEG C of reactions are added by the amount of 1:8000
30min, every hole are cleaned with 300 μ L PBST, and totally 5 times.The every 100 μ L of hole of TMB developing solution, color development at room temperature 15-30min, 1M sulfuric acid
100 holes μ L/, color development stopping observe result.It selects positive colony and is subcloned to character and stablized, as engineering cell strain standard
Standby amplification culture.
7. the preparation and purification of the ascites of anti-m6A-RNA antibody
Expand culture using the positive cell after clone, through BALB/c mouse, 10-15 days harvest ascites is injected intraperitoneally.It adopts
The anti-m6A-RNA monoclonal antibody purified in ascites with octanoic acid-ammonium sulfate salting-out process.With the filtering with microporous membrane in 0.45 μm of aperture
Ascites.The sodium-acetate buffer of 2 parts of 0.06mol/L, pH4.0 are added in 1 part of ascites, with 0.1mol/L HCl tune pH to 4.5.Room
Temperature stirs in lower 30min and is slowly added to octanoic acid dropwise, and 33 μ l/ml ascites, 4 DEG C of standing 2h are added；4 DEG C of 6 000 × g of low temperature centrifugations
30min abandons precipitating, and supernatant is filtered through sand core funnel, and 0.277g/ml ammonium sulfate is added in 30min under ice bath, keeps ammonium sulfate full
With degree about 45%, 1h or more is stood；4 DEG C of 6 000 × g of low temperature are centrifuged 30min, abandon supernatant, and precipitating is dissolved in appropriate PBS solution
In, 4 DEG C of dialysed overnights.Protein A affinity column, adsorbing separation, albumen needed for retaining are used when secondarily purified, and are carried out
The purity that 12%SDS-PAGE analyzes and identifies extraction monoclonal antibody reaches 98% or more.
The technical solution of embodiment 1,2 can also prepare the antibody of anti-methylate DNA and the antibody of anti-methylation RNA.Its
In, the antibody of anti-methylate DNA includes but is not limited to anti-5- methyl born of the same parents phonetic (5-mC), anti-N6 methyl purine (N6-mA), anti-7- first
Base guanine (7-mG)；The antibody of anti-methylation RNA includes but is not limited to anti-N6- methyl adenine, anti-1-methyladenosine, anti-7-
The spy of methylguanosine, anti-5- methylcytidine, anti-N2- dimethylguanosine, anti-5- (methylol) cytidine, anti-N- acetylcytosine nucleosides
Heterogenetic antibody.
Embodiment 3
Amino magnetic bead coupled antibody
1. balancing EDC and NHS to room temperature.
2. adding the NHS of the EDC and 0.6mg of 0.4mg in the antibody that 1mL concentration is 1mg/mL, 15min is reacted at room temperature.
3. adding 1.4 μ L beta -mercaptoethanols.
4. adding amino magnetic bead 1ml, 2h is reacted at room temperature.
5. the glycine of final concentration of 50mM is added to terminate reaction.
6. after magnetic-adsorption magnetic bead, being washed 3 times with PBS.The PBS of the last magnetic bead Sodium azide for being added to 0.1% is resuspended,
And it is saved at a temperature of 4 DEG C
Embodiment 4
Carboxyl magnetic bead coupled antibody
1. pipette in the carboxyl magnetic bead to 15mL tip centrifuge tube of 0.5mL (10mg), and be placed on Magneto separate frame until
After supernatant becomes completely thorough, abandoning supernatant is carefully moved with suction pipe.
2. the MES buffer of 5mL is added to mix well washing.Centrifuge tube is placed on Magneto separate frame until supernatant becomes clear
Afterwards.It is carefully moved with suction pipe and abandons supernatant.
3. repeating 2 three times.After last time is washed, magnetic bead is resuspended in the MES buffer of 5mL.
4. EDAC is placed in room temperature 30 minutes from refrigeration place taking-up.EDAC needed for accurately weighing 1.6mg, which is added, is equipped with magnetic
Acutely oscillation shakes up in the centrifuge tube of pearl.
5. centrifuge tube at room temperature, is placed in priming reaction 30min on rotation blending instrument.In reaction process, pay attention to not allowing magnetic
Pearl precipitating is built up together.
6. centrifuge tube is placed on Magneto separate frame after supernatant becomes clear.It is carefully moved with suction pipe and abandons supernatant.With 5mL MES
Buffer washs four times.
7. adding the antibody after purification of 200 μ g into suspension containing magnetic beads, acutely concussion is mixed.Centrifuge tube is placed at room temperature
Rotate coupling reaction 16h on blending instrument.
8. centrifuge tube is placed on Magneto separate frame after supernatant becomes clear.It is carefully moved with suction pipe and abandons supernatant.With 5mL MES
Buffer washs 4 times.
9. be added 1M/L glycine 5mL, room temperature in rotation blending instrument on react 30min.
10. centrifuge tube is placed on Magneto separate frame after supernatant becomes clear.It is carefully moved with suction pipe and abandons supernatant.Use 5mL
After MES buffer washs 4 times, magnetic bead is deposited in into 4 DEG C of preservations.
It should be appreciated that antibody described in embodiment 3,4 is anti-including monoclonal antibody after purification or mostly.
Embodiment 5
Fibroin protein film is coupled anti-RNA antibody
1. using 0.05mol/L, the glutaraldehyde that W/V ratio is 25% is diluted to 1% by pH9.5 carbonate buffer solution (CBS).
2. 2h is impregnated in 37 DEG C of water-baths by fibroin protein film 1% glutaraldehyde of 50ml.
3. step 2 gained mixture to be shaken at a temperature of 4 DEG C to washing 30min with the PBST of 50mL, concussion washing is repeated,
Concussion washing 3 times in total.
4. preparing the anti-RNA monoclonal antibody leaching of resulting mouse by 2 technical solution of embodiment with final concentration of 50 μ g/mL
Steep fibroin protein film, 4 DEG C of soaked overnights.It can be by anti-RNA antibody coupling on fibroin protein film or other biological material.
5. 50ml 0.2mol/L lysine is added, to close remaining aldehyde radical, reaction is terminated.
30min is washed in 4 DEG C of concussions of 6.PBST, totally 3 times, is discarded remaining termination reagent, is saved at 4 DEG C stand-by.
Embodiment 3,4 is the specific embodiment of magnetic bead of the invention and antibody coupling step, and embodiment 5 is silk of the invention
The specific embodiment of fibroin film and antibody coupling step.
It should be appreciated that the step of magnetic bead or fibroin protein film and antibody coupling, can't should be limited in the above tool
Content described in body embodiment.
It should be appreciated that the coupling step of the antibody and extraction material for nucleic acid of the invention can be using arbitrary existing
There is technical solution.Wherein, for conjugate, that is, nucleic acid extraction of the present invention material of the antibody of nucleic acid and extraction material.
Embodiment 6
Immunomagnetic beads method extracts peripheral blood dissociative DNA
1. simulating clinical sample
A certain amount of inhuman source DNA of small fragment DNA is put into human normal plasma to simulate height in cancer patient's blood plasma
The peripheral blood dissociative DNA of fragmentation, the simulating blood plasma contain 3 kinds of DNA fragmentation mixtures, length be respectively 102bp, 268bp and
The final concentration of 100ng/mL blood plasma of 402bp, DNA.
2. immunomagnetic beads method extracts peripheral blood dissociative DNA
500 μ L of simulating blood plasma is added in 2mL EP pipe, is added and is coupled the mostly anti-50 μ L of magnetic bead of anti-DNA, after mixing
It is incubated at room temperature 10min；Magneto separate abandons liquid；It is reverse for several times that 1mL cleaning solution I (PBS contain, 1% polysorbas20) is added, Magneto separate,
Abandon liquid；1mL cleaning solution II (Tris-HCL of 10mM, the EDTA, pH8.0 that concentration is 1mM) is added, overturns for several times, Magneto separate,
Abandon liquid；100 μ L eluents (20mM Tris, pH 7.5,300mM NaOAc, 2mM EDTA, pH 8,0.25%SDS) is added
It is eluted, after mixing, is being placed at room temperature for 2min；Magneto separate, supernatant be extract peripheral blood dissociative DNA, the concentration of DNA with
Purity is with NanoDrop spectrophotometer measurement and records.
It should be appreciated that the anti-DNA of coupling of embodiment 6 mostly anti-magnetic bead is prepared using according to 1 technical solution of embodiment
Resulting DNA is mostly anti-to be prepared according to the coupling method of embodiment 3 or 4.
Embodiment 6 is the tool of the nucleic acid extraction material extraction nucleic acid of the invention obtained using embodiment 3,4 coupling steps
Body embodiment, the specific steps are as follows:
It extracts: the blood plasma containing DNA being extracted with the nucleic acid extraction material, obtain the how anti-coupling magnetic of the anti-DNA of DNA-
Pearl compound；
Washing: it is successively washed with cleaning solution I, cleaning solution II, the how anti-coupling magnetic of the anti-DNA of the DNA- for the impurity that is removed
Pearl compound；
Elution: using elution, extracts target dna needed for obtaining.
Wherein, the nucleic acid extraction material is the conjugate of mostly anti-and by chemical group modification the magnetic bead of anti-DNA；
The chemical group for modifying magnetic bead includes but is not limited to amino, carboxyl, phenyl, phenolic group, sulfydryl, hydroxyl, imidazole radicals, Yin
Diindyl base, guanidine radicals.
Embodiment 7
Fibroin protein film is coupled the total serum IgE that RNA specific polyclonal antibody centrifugal column method extracts breast cancer cell
As shown in figure 4, being the flow diagram of the present embodiment, specific step is as follows for the present embodiment:
1. centrifugal column filling material is to be coupled RNA specific polyclonal using what the technical side of embodiment 6 was prepared
Antibody fibroin protein film.
2. by the breast cancer cell (1x10 by being obtained after selected by flow cytometry apoptosis3It is a) it is added in the EP pipe of 2mL, then
Be added isometric lysate (Proteinase K of the 10mg/mL of 30 μ l, the guanidine thiocyanate of 5.6M, the EDTA of 100mM, 20mM's
Tris, 0.01% Triton X-100), it overturns oscillation and mixes, react at room temperature 2min.Then 8000g is centrifuged 5min.
3. step 2 gained supernatant is added in centrifugal column, 10000g is centrifuged 2min, removes unbonded impurity.
4. cleaning solution I (PBS contains 1% polysorbas20) 700 μ L, 10000g is added into the centrifugal column of step 3 is centrifuged 1min,
Abandon the liquid in collecting pipe.
5. add cleaning solution II (Tris-HCL of 10mM, the EDTA, pH 8.0 that concentration is 1mM) 700 μ L, 10000g from
Heart 1min abandons the liquid in collecting pipe.
6. eluent (20mM Tris, pH 7.5,300mM NaOAc, 2mM EDTA, pH 8,0.25%SDS) 50 μ are added
In on film, the total serum IgE eluted on incubation at room temperature 2-5min, 10000g centrifugation 5min collection membrane measures l through NanoDrop,
The concentration of RNA is that the purity of 2ng/ μ L, RNA see absorption photometric ratio under 260nm and 280nm, OD260/OD280 2.05.
Embodiment 7 is the specific of the nucleic acid extraction material extraction nucleic acid of the invention obtained using 5 coupling step of embodiment
Embodiment, the specific steps are as follows:
It extracts: the breast cancer cell containing RNA being extracted with the nucleic acid extraction material, obtain RNA-RNA specificity
Polyclonal antibody is coupled fibroin albumen membrane complex；
Washing: it is successively washed with cleaning solution I, cleaning solution II, the RNA-RNA specific polyclonal for the impurity that is removed
Antibody coupling fibroin albumen membrane complex；
Elution: using elution, extracts target RNA needed for obtaining.
Wherein, the nucleic acid extraction material is the conjugate of anti-RNA specific polyclonal antibody and fibroin protein film.
Embodiment 8
Cyanogen bromide-activated agarose microbeads particle is coupled anti-DNA single-chain antibody and extracts a large amount of DNA for extracting calf thymus
As shown in figure 5, being the flow diagram of the present embodiment, 5 in Fig. 5 indicate cyanogen bromide-activated agarose microbeads particle
It is coupled anti-DNA single-chain antibody, specific step is as follows for the present embodiment:
The agarose microbeads particle of sufficiently preswollen cyanogen bromide-activated is added in 1.10mL filled column.
2. using 0.1mol/L NaHCO3Solution, 8.2 repeated flushing pillar of pH.
3. it is anti-that the anti-DNA monoclonal that 0.5mol/L NaCl and 0.2g is prepared according to the technical solution of embodiment 2 is added
Body.Suspension shaken at room temperature 3h or 4 DEG C are overnight.
4. 5ml 1mol/L amion acetic acid is added to close residual activity group.
5. rinsing gel, then the di(2-ethylhexyl)phosphate with 0.1mol/L pH7.6 with 4.0 sodium acetate-acetic acid of 0.1mol/L pH first
Hydrogen potassium rinses pillar.Then steady to OD260 baseline with PBS flushing pillar, zeroing.
6. by calf thymus cell through lysate (Proteinase K of the 10mg/mL of 30 μ l, the guanidine thiocyanate of 5.6M, 100mM
EDTA, the Tris of 20mM, 0.01% Triton X-100) cracking after sample flow add through above-mentioned filler, can repeatedly on
Sample 1-2 times.
7. adding cleaning solution (PBS contains 1% polysorbas20) 3-5 column volume, washing to OD260 value is zero.
8. adding eluent (20mM Tris, pH 7.5,300mM NaOAc, 2mM EDTA, pH 8,0.25%SDS), Bian Jia
Side monitors OD260, collects peak value sample, the DNA of the calf thymus purified.
Embodiment 8 is a kind of specific embodiment of the method for extraction nucleic acid of the invention, the specific steps are as follows:
Coupling: anti-DNA single-chain antibody and cyanogen bromide-activated agarose microbeads particle are coupled, and obtain nucleic acid extraction material
Material；
It extracts: the sample after calf thymus cell cracking being extracted with the nucleic acid extraction material, obtain the anti-DNA of DNA-
Single-chain antibody is coupled cyanogen bromide-activated agarose microbeads particle composites；
Washing: being washed with cleaning solution, and the anti-DNA single-chain antibody of the DNA- for the impurity that is removed is coupled cyanogen bromide-activated fine jade
Lipolysaccharide microsphere compound；
Elution: using elution, extracts target dna needed for obtaining.
Wherein, the nucleic acid extraction material is that anti-DNA single-chain antibody and cyanogen bromide-activated are coupled agarose microbeads particle
Conjugate.
Embodiment 9
The mode (high-temperature elution) of silicon substrate micro-fluidic chip extraction methylate DNA
As shown in fig. 6, being the structural schematic diagram of silicon base chip 6.Sample to be extracted is passed through by well 7, into miniflow
Behind road 8, it is coated with area 9 by anti-methylate DNA, methylate DNA and the specific binding of anti-methylate DNA in sample to be extracted,
To be bonded to coating area 9, after washed liquid washs impurity, is heated using heating module 11 and carry out high-temperature elution, hot wash
De- DNA flows to methylate DNA collection channel 10, and then collects extracted methylate DNA.
Specific step is as follows:
1. in ethyl alcohol and deionized water be cleaned by ultrasonic silicon wafer 5min, then place it in volume ratio be 7:3 the concentrated sulfuric acid and
Silicon face 30min is handled in the mixed liquor of hydrogen peroxide, after with deionized water flushing pipe and with being dried with nitrogen.
2. silicon wafer is immersed to the ethanol solution arrest reaction 30min of the 3- aminopropyl triethoxysilane of 10mmol/L again,
After with ethyl alcohol rinse twice and with being dried with nitrogen, silicon wafer is placed in 45min in 100 DEG C of baking ovens.
3. silicon wafer is immersed the terephthalaldehyde acetone soln of 10mmol/L and stands 30min.
4. by the antibody of the resulting anti-methylate DNA of embodiment 1 with the direct point of the PBS buffer solution point sample instrument of 50 μ g/mL
In the silicon chip surface modified, entire silicon wafer places 4h under 37 DEG C of constant-temperature constant-humidity environments with sessile antibody, is then placed within
Each 5min of constant temperature oscillation in 0.5%Tween-20PBS solution and PBS buffer solution.
5. it is other unbonded that silicon wafer is placed to 30min closing in 1mol/L trishydroxymethylaminomethane (pH 7.4) solution
Aldehyde groups.
6. taking 100 μ L to be added to the sample introduction end of micro-fluidic chip for containing the solution sample in need for extracting DNA.By micro-
Amount syringe pump slowly control sample flow through antibody coating area, control the steering of pump so that sample it is repeated multiple times pass through antibody
It is coated with area.
7. 200 μ L cleaning solutions (PBS contains 1% polysorbas20) are added by micro-fluidic chip injection port, wash 3 times.
8. 50 μ L TE buffers are added, stream adds to the combined area antibody-DNA, using beneath chips heating module by antibody-
The combined area DNA is heated to 90-95 DEG C, is incubated for 2-5min.Syringe pump is controlled, the DNA that hot wash takes off is flowed into downstream working region.
Embodiment 9 is a kind of specific embodiment of the method for extraction nucleic acid of the invention, the specific steps are as follows:
Coupling: anti-methylate DNA and silicon wafer are coupled, and obtain nucleic acid extraction material；
It extracts: the sample containing methylate DNA being extracted with the nucleic acid extraction material, obtain the anti-methyl of methylate DNA-
Change DNA silicon wafer compound；
Washing: it is washed with cleaning solution, the anti-methylate DNA silicon wafer compound of the methylate DNA-for the impurity that is removed；
Elution: using elution, extracts target dna needed for obtaining.
Wherein, the nucleic acid extraction material is the conjugate of anti-methylate DNA and silicon wafer.
Embodiment 1-9 be specific embodiments of the present invention, it should be understood that these being merely to illustrate property of embodiment purposes and
It is understood not to limit the invention in any way.
Comparative example 1
Silicon substrate paramagnetic particle method extracts peripheral blood dissociative DNA
1. simulating clinical sample
A certain amount of inhuman source DNA of small fragment DNA is put into human normal plasma to simulate height in cancer patient's blood plasma
The peripheral blood dissociative DNA of fragmentation, the simulating blood plasma contain 3 kinds of DNA fragmentation mixtures, length be respectively 102bp, 268bp and
The final concentration of 100ng/mL blood plasma of 402bp, DNA.
2. the extraction step of silicon substrate paramagnetic particle method
It is operated by certain domestic brand kit specification: 500 μ L of simulating blood plasma being added in the EP pipe of 2mL, 500 μ L are added
Buffer LB1 (Proteinase K of the 10mg/mL of 30 μ L, the guanidine thiocyanate of 5.6M, the Tris of the EDTA of 100mM, 20mM,
0.01% Triton X-100), after mixing, it is placed in 56 DEG C of water-bath and is incubated for 30min.Centrifuge tube is taken out and is added
Isometric isopropanol and 50 μ L silicon substrate magnetic beads, are mixed by inversion for several times, Magneto separate, abandon waste liquid；The Buffer WB I (4M of 1mL is added
Guanidine hydrochloride, 25% ethyl alcohol and the EDTA of 10mM), be mixed by inversion, Magneto separate, abandon waste liquid；The Buffer WB II of 1mL is added
(70% ethyl alcohol), is mixed by inversion, Magneto separate, abandons waste liquid；It is primary to repeat Buffer WB II (70% ethyl alcohol)；It opens at room temperature
Lid stands 5min；Elution Buffer (Tris-HCL of 10mM, the EDTA, pH that concentration is 1mM of 100 μ L is added
8.0) it after, mixing completely, is placed in water-bath, is incubated for 10min；Magneto separate, supernatant are the peripheral blood dissociative DNA extracted, DNA
Concentration and purity with NanoDrop spectrophotometer measurement and record.
Embodiment 10
The comparison result that the immunomagnetic beads method and 1 silicon substrate paramagnetic particle method of comparative example of embodiment 6 extract peripheral blood dissociative DNA joins table
Three.
Table three
As shown in fig. 7, Fig. 7 is the agarose gel electrophoresis figure of embodiment 6 and comparative example 1, wherein M indicates DL in Fig. 7
2000 DNA maker, 1 indicates that embodiment 6 extracts peripheral blood dissociative DNA, and 2 indicate that the silicon substrate paramagnetic particle method extraction of comparative example 1 is outer
All blood dissociative DNAs.After combining Fig. 7 it is found that the magnetic bead of embodiment 6 has been coupled the antibody for target dna by table three, nucleic acid is obtained
Extract material, further extract target dna, the rate of recovery of DNA considerably beyond on the market at present used in silicon substrate paramagnetic particle method
Extract the rate of recovery of peripheral blood dissociative DNA；The DNA concentration that embodiment 6 is extracted is higher than silicon substrate paramagnetic particle method and extracts peripheral blood dissociative DNA
Concentration.
The present invention can be coupled described by using collagen, silk, cellulose, magnetic corpusculum, chitosan, silica etc.
Antibody for target nucleic acid or microporous barrier, microballoon, magnetic bead, fiberfill, the porous media prepared to it carry out certain change
It after learning base group modification (such as amino, carboxyl, phenyl), then is coupled with for the antibody of target nucleic acid, obtains nucleic acid extraction
Material.A kind of nucleic acid extraction medium provided by the invention includes present invention nucleic acid extraction material as described above.The present invention provides
A kind of method for extracting nucleic acid, Extraction medium, kit, can be with the nucleic acid interaction in sample to be extracted, can be reversible
It forms nucleic acid-nucleic acid and extracts material composite, and then can be used for extracting nucleic acid.
Nucleic acid is extracted using the catching method of immune antiboidy specificity, specificity, can be captured with high efficiency and accurately
To corresponding nucleic, the rate of recovery that nucleic acids in samples to be processed extracts is improved, and is can reduce needed for extracting nucleic acid to a certain extent
Time, improve extraction efficiency.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown here.
Claims (10)
1. a kind of method for extracting nucleic acid, which comprises the steps of:
Coupling will be coupled for the antibody of target nucleic acid with material is extracted, and obtain nucleic acid extraction material；
It extracts, sample to be extracted is added in the nucleic acid extraction material, in the nucleic acid extraction material and sample to be extracted
Target nucleic acid specific binding, obtain nucleic acid-nucleic acid extract material composite；
Washing washs the nucleic acid-nucleic acid extraction material composite with cleaning solution, is mentioned with the nucleic acid-nucleic acid for the impurity that is removed
It draws materials compound；
The nucleic acid-nucleic acid after eluting, will be washed extracts material composite and elutes, to obtain target nucleic acid molecules.
2. the method according to claim 1 for extracting nucleic acid, which is characterized in that the antibody for target nucleic acid includes
Anti-DNA antibody, anti-RNA antibody.
3. the method according to claim 1 for extracting nucleic acid, which is characterized in that the antibody for target nucleic acid includes
Monoclonal antibody, polyclonal antibody, Antibody Fab fragment, single-chain antibody, single domain antibody.
4. the method according to claim 1 for extracting nucleic acid, which is characterized in that the nucleic acid includes methylate DNA, methyl
Change RNA；The antibody for target nucleic acid includes the antibody of the antibody of anti-methylate DNA, anti-methylation RNA.
5. the method according to claim 4 for extracting nucleic acid, which is characterized in that the antibody packet of the anti-methylate DNA
Include phonetic anti-5- methyl born of the same parents, anti-N6 methyl purine, anti-7- methyl guanine；The antibody of the anti-methylation RNA includes anti-N6- first
Base adenine, anti-1-methyladenosine, anti-7- methylguanosine, anti-5- methylcytidine, anti-N2- dimethylguanosine, anti-5- (methylol)
Cytidine, anti-N- acetylcytosine nucleosides.
6. a kind of nucleic acid extraction medium, which is characterized in that mentioned including the nucleic acid as made from coupling step in claim 1
It draws materials.
7. nucleic acid extraction medium according to claim 6, which is characterized in that the nucleic acid extraction material be it is natural or
By chemical group modification can be with the medium of the antibody coupling for target nucleic acid；The chemical group includes ammonia
Base, carboxyl, phenyl, phenolic group, sulfydryl, hydroxyl, imidazole radicals, indyl, guanidine radicals.
8. nucleic acid extraction medium according to claim 6, which is characterized in that the material of the nucleic acid extraction material includes glue
Original, silk, cellulose, magnetic corpusculum, chitosan, silica.
9. nucleic acid extraction medium according to claim 8, which is characterized in that the presentation knot of the nucleic acid extraction Material texture
Structure includes microporous barrier, microballoon, fiberfill, porous media, silicon substrate micro-fluidic chip.
10. a kind of kit for extracting nucleic acid, which is characterized in that including the nucleic acid as described in claim 5-9 any one
Extraction medium, cleaning solution, eluent.
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