CN107119039A - It is a kind of to organize not grinding the method for directly extracting nucleic acid - Google Patents
It is a kind of to organize not grinding the method for directly extracting nucleic acid Download PDFInfo
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- CN107119039A CN107119039A CN201610099919.XA CN201610099919A CN107119039A CN 107119039 A CN107119039 A CN 107119039A CN 201610099919 A CN201610099919 A CN 201610099919A CN 107119039 A CN107119039 A CN 107119039A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
It is more particularly to a kind of to organize not grinding the method for directly extracting nucleic acid the present invention relates to nucleic acid extraction field.Lengthy and tedious in order to solve to operate in traditional nucleic acid extraction process, extracting cycle is long, it is impossible to realize automation, contact the defects such as toxic reagent, and in novel nucleic acids extraction apparatus extraction process, animal tissue's sample need to grind cracking centrifugation in advance, it is impossible to the technical problem that directly upper machine is extracted.The present invention provides a kind of method organized not grinding and directly extract nucleic acid, and using unique lytic reagent, supporting 96 passage instrument for extracting nucleic acid realizes the integration of instrument consumptive material, the full-automatic nucleic acid for extracting animal tissue.The reagent is the lysate after optimizing for different samples, and the instrument is instrument for extracting nucleic acid, can optimize extraction conditions according to different tissue samples.The features such as present invention has that step is simple, full-automatic operation, nucleic acid purity are high, extracting cycle is short, does not contact toxic reagent.
Description
Technical field
The invention belongs to nucleic acid extraction technical field, it is related to method for extracting nucleic acid, more particularly to organizes not grinding the method for directly extracting nucleic acid.
Background technology
Nucleic acids research is the important topic in modern biology, medical research.Nucleic acid as hereditary information carrier, it is the material base of gene expression, in addition to normally growing, developing and breeding in organism etc. and had a very important role in vital movement, its abnormal conditions with life, such as tumour generation, radiation insult, genetic disease also have substantial connection.Sweating is told with the technology of molecule life in recent years, is hybridized using nucleic acid, the molecule diagnosis and detection technique of nucleic acid amplification and nucleic acid sequence analysis as representative increasingly highlight vital effect in numerous areas.New molecular Biological Detection technology includes PCR (PCR), microchip, high-flux sequence etc., but, all modern molecular biology detection techniques problems faced first is how that the Nucleic acid quality and its integrality that quickly and effectively separate and extract from complicated and diversified biological specimen after required genomic nucleic acids, and extracting can all directly influence subsequent experimental result.At present, global researcher also wicked many breakthrough progress on the technical method of nucleic acid separation and Extraction.Since being found first by the mankind from 1869, many researchs have just carried out unremitting exploration in the extracting method of nucleic acid, the various materials and reagent of nucleic acid extraction are improved, the various chemical reagent such as dodecyl sodium sulfate, phenol, urea, guanidine hydrochloride are applied in nucleic acid extraction experiment one after another.Research with milestone significance finds to include during this：1948, Chargaff Eng people obtained the DNA with heaven and earth shape using sodium citrate and RNase, and nineteen fifty-seven Kirby extracts DNA with phenol method, i.e., isometric phenol is added into cell pyrolysis liquid：Chloroform：Isoamyl alcohol (25:24:1 volume) mixed liquor, collected after centrifugation upper strata aqueous phase and with isopropanol precipitating nucleic acid therein after rinsing salinity through ethanol, uses the buffer solutions such as TE or distilled water dissolving to prepare DNA.Mamur in 1961 creates dodecyl sodium sulfate (SDS) method, it is used to extract bacteria plasmid DNA earliest, the principle of the method is to keep carrying out the alkaline denaturation of selectivity to the bacterial chromosomal dna of high relative molecular mass while double-strand based on covalently closed circular DNA, it is allowed to and bacterioprotein and broken cell membrane formation compound of the surface covered with dodecane sulfonate, it is denatured material after centrifugation to be removed, DNA just can be reclaimed from supernatant.Researchs of the Chirgwin in 1979 to forefathers has carried out the summary of system, found guanidine thiocyanate method and extract RNA, a kind of strong protein denaturant during guanidinium isothiocyanate, both rupturable cell, the effect of nuclease can also be suppressed, but the operation of this method is relatively complicated, therefore Chomczynski and Sacchi in 1987 has started one-step method and has extracted RNA on this basis, improves the speed of RNA extractions and is therefore widely used.Serve, about nucleic acid extracting method still in constantly Improvement.Opened within 90 years from 20th century, the need for adapting to modern molecular biology detection experiment high flux, high sensitivity, automation mechanized operation, the method for extracting nucleic acid with magnetic bead is arisen at the historic moment, this method is the perfect adaptation of nanosecond science and technology and biotechnology, with the incomparable advantage of other method for extracting nucleic acid, have the advantages that high flux operation, short used time simple to operate, safety non-toxic and purity high concentration are big.
Nucleic acid extraction is the starting point of downstream nucleic acid detection, research or product development, and the quality and integrality of separated nucleic acid directly affect research or diagnostic result, therefore one of method of molecular biology most critical during nucleic acid extraction.Generally, successful nucleic acid isolates and purifies four important steps of needs：Tissue or cell broken and crack, and the complex of albumen deformation, the inactivation of nuclease and the removal of pollutant.Preferable method for extracting can obtain high-quality target nucleic acid, while without albumen, sugar, fat and other pollution of nucleic acid.Many specialized nucleic acid extraction methods existing at present can extract DNA, RNA or total nucleic acid from various biological samples.Most of these methods have been split into commercialized kit, and what is had can be implemented in combination with automation extracting with instrument, so that extractive process is greatly simplified.
Nucleic acid extraction method mainly includes：Alkali density method, phenol chlorine method, high salt precipitation method, CTAB extraction process, silicon dielectric posts method, ion-exchange, Magnet Treatment etc..
Alkaline lysis is used for isolated plasmid dna and Escherichia coli, in the case of dodecyl sodium sulfate presence, and the method can effectively handle the bacterial cultures of all coli strain and volume in 1ml to more than 500ml.The principle of the method is the alkaline denaturation for keeping carrying out HMW chromosomal DNA while double-strand selectivity based on covalently closed circular DNA；Chromosomal DNA formation big compound of the surface covered with dodecane sulfonate of bacterioprotein, broken cell membrane and denaturation；Through centrifugation, the material back of the body of these denaturation is removed, and DNA just can be reclaimed from supernatant.
Phenol-chloroform extraction process is a classical way of nucleic acid separation.Centrifugation after cell lysis separates the aqueous phase containing nucleic acid, adds isometric phenol：Chloroform：Isoamyl alcohol (25:24:1 volume) mixed liquor, collected after centrifugation upper strata aqueous phase and with isopropanol precipitating DNA therein, ethanol is rinsed and discarded after salinity, TE buffer solutions or distilled water dissolving target DNA.
Guanidinium isothiocyanate is a kind of strong protein denaturant, can both rupture cell, can also suppress the effect of nuclease.Guanidinium isothiocyanate is used for RNA extractings and proposed first by Ulrich etc. (1977).The method is comparatively laborious, and the guanidinium isothiocyanate invented by Chomczynski and Sacchi (1987)-phenol-chloroform extracting one-step method is replaced.One-step method is to realize separating for RNA and DNA with the acid solution extracting comprising guanidinium isothiocyanate, acetate, phenol and chloroform, finally reclaims the total serum IgE in aqueous phase with isopropanol precipitating.
Using RNA and DNA in electrolytic solution different solubility, the two is separated, conventional method is extracted with 1M common salts, obtained DNP mucus is swayed together with the chloroform containing a small amount of octanol, make emulsification, then be centrifuged off protein, now protein gel is rested in the middle of aqueous phase and chloroform phase, and DNA is located in upper strata aqueous phase, DNA sodium salts can be precipitated out with 2 times of ethanol of volume 95%.
CTAB is a kind of nonionic detergent, can from low ionic strength solution precipitate nucleic acids and acidic polysaccharose, and protein and neutral polysaccharide are remained in solution.In high inonic strength solution, CTAB then will not precipitate nucleic acids, but with albumen formation compound.Therefore, CTAB is usually used in the purification of nucleic acid from the organism that can produce a large amount of polysaccharide, such as plant and some Gram-negative bacterias.The subsequent step of this method is also to go isolating protein, the salt of pollution and other materials to carry out purification of nucleic acid by using organic solvent, ethanol precipitation etc..
Silicon dielectric posts method can be used for manual extraction, it can also be used to which automation is extracted.Its principle is as shown in Figure 8.
Magnet Treatment is generally used for automation and extracted, biomagnetic beads are a kind of new functionalization solid carriers, the active group of its pan coating, it can be coupled with various bioactivators, the features such as mobility with liquid and solid magnetic material, can be with displacement and concentration in the presence of external magnetic field, after external magnetic field is removed, slightly vibrating or aspirating can be dispersed in liquid again, so that the separation of solid liquid phase become it is very efficient and convenient, can obtain purity very high targeting substance by simply eluting.
Many well-known biological reagent companies have developed various nucleic acid extraction kits based on these traditional method for extracting nucleic acid, for DNA and RNA to be separated and extracted from various a variety of tissue samples, Fig. 9 is shown in different method for extracting nucleic acid contrasts.But, the operation such as precipitation and centrifugation included in traditional nucleic acid extraction technology needs to use substantial amounts of biological specimen, and traditional extraction technology step is more numerous and diverse, time-consuming length, yield is low, automation mechanized operation difficult to realize, most of method also needs to operating personnel and directly contacts poisonous chemical reagent.And it is big to centrifuge column type method sample requirements, expend sample more, for some rare samples, it is applied by great limitation, centrifugal column method is extracted accounting needs and is centrifuged repeatedly simultaneously, it is not easy to high flux, automation mechanized operation, particularly in fields such as gene diagnosis, the monitoring and controls of SARS Epidemic, extracting nucleic acid using centrifugal column method needs substantial amounts of operating personnel and instrument and equipment to meet demand.And although new nucleic acid extraction technology disclosure satisfy that high flux and automation mechanized operation, but to some animal vegetable tissues, hand-ground cracking process in traditional nucleic acid extraction process still can not be broken away from, it is impossible to realize the full-automatic nucleic acid extraction of tissue samples, so that nucleic acid extraction becomes cumbersome, the cycle is elongated.
The content of the invention
The present invention needs to overcome the defect in traditional nucleic acid extraction process：
1st, step is numerous and diverse, and time-consuming length, yield is low, is difficult to realize automation mechanized operation, need to contact toxic chemical；
2nd, centrifugation column type method sample requirements are big, limited to for rare sample, it is necessary to be centrifuged repeatedly, it is impossible to realize high flux and automation；
3rd, new nucleic acid extraction technology still can not break away from hand-ground cracking process in traditional nucleic acid extraction process, it is impossible to realize the full-automatic nucleic acid extraction of tissue samples so that nucleic acid extraction becomes cumbersome to some animal vegetable tissues, and the cycle is elongated；
The present invention extracts nucleic acid using paramagnetic particle method, is solved the above problems by optimizing lysate and supporting 96 passage Full automatic instrument for extracting nucleic acid, realizes that tissue is not ground and directly extracts nucleic acid.
The concrete technical scheme of lysate is, the composition of basic lysate is guanidine hydrochloride, guanidinium isothiocyanate, Tris and Qula are logical, wherein guanidine hydrochloride and guanidinium isothiocyanate are strong protein denaturant, for rupturing tissue, the protein ingredient of cell membrane, help intracellular nucleic acid release, there is provided high chaotropic salt environment simultaneously, change the arrangement mode of hydrone in solution, make to expose by the nucleic acid that large quantity of moisture attached bag is wrapped up in, magnetic bead is wrapped with the film of silicidation, under lysate environment, hydroxyl on the pentose of exposed nucleic acid can be adsorbed with the suicide material outside magnetic bead, relatively stable compound is formed to combine on magnetic bead.According to different samples, on the basis of basic lysate, matched by adjusting each constituent concentration of basic lysate, and increase other adjuvants, including SLS, SDS, urea, ethanol, CTAB and PVPP etc..In sample processes are extracted, according to the difference of sample cell wall or the composition the Nomenclature Composition and Structure of Complexes of outer wall, lysozyme, Proteinase K etc. is added in cracking process to help to crack, β -1 between the peptide glycan for the cracking gram-positive bacteria cell wall that wherein lysozyme can be specifically, 4 glycosidic bonds, accelerate the cracking of gram-positive bacteria.Proteinase K be it is a kind of be the wider serine protease of cleavage activity.It cuts protein molecule in the carboxy-terminal peptide bond of aliphatic amino acid and aromatic amino acid, can fully degrade tissue or cell, the nucleic acid in sample is fully discharged, while can add nucleic acid settling agent to help nucleic acid to settle compared with small sample for sedimentation coefficient.
The concrete technical scheme of 96 passage instrument for extracting nucleic acid is, 96 passage instrument for extracting nucleic acid use Beads enrichment technology, its principle is the nanoscale magnetic bead of silicidation, absorption nucleic acid that can be special under the conditions of high chaotropic salt, elute nucleic acid under low-salt conditions, magnetic bead can be adsorbed with magnetic bar magnet in itself, therefore after the nucleic acid that is discharged after cracking of sample is adsorbed through magnetic bead, nucleic acid transfer operation is completed by bar magnet, the process of rinsing decontamination and elution nucleic acid is completed in different liquid respectively.When cracking different samples, the complexity cracked according to sample, speed and the time of cleavage step of bar magnet and bar magnet set vertical tremor are set during program editing in parameter setting, 96 instrument for extracting nucleic acid are provided with semiconductor heating refrigeration module simultaneously, cracking process can heat lysate, the preference temperature that the enzymes such as Proteinase K play activity is provided, to adapt to the cracking of different tissues sample.
96 passage instrument for extracting nucleic acid are the high-tech products using advanced Beads enrichment technology, high with automaticity, and extraction rate is fast, as a result stablizes, advantage easy to operate.All purification procedures of 96 passage instrument for extracting nucleic acid are completed in 96 deep-well plates, have been completely free of the step such as required centrifugation, filtering in traditional extraction, purifying.The system can complete the rapid extraction purifying of 1-96 original samples samples such as (including) blood, tissue, cell, secretion, bacterium, plant, legal medical expert's sample and amplified productions in 30-60 minutes simultaneously.The high-quality nucleic acid (DNA/RNA) extracted can be used for highly sensitive downstream analysis, such as quantitative PCR, clinical molecular diagnosis, gene expression analysis, genetic analysis, legal medical expert and infectious disease research；Nucleic acid after purification may be directly applied to digestion, identification and the medical diagnosis on disease of next step, treatment etc..Other supporting professional paramagnetic particle method kits, it is possible to achieve automatically complete the purification of protein or polypeptide fragment, the separation and concentration of specific bacterium, cell and various tumor markerses etc..Producer's kit is comprehensive, and non-anticoagulation (solidification blood) can directly extract genomic DNA, it is not necessary to any pretreatment；Animal tissue is not required to grinding digestion in advance, tissue block can directly upper machine extraction DNA.
Advantages of the present invention has the following aspects：
1st, high flux operation can be realized and automated, extraction time with a sample is that the processing to 96 samples can be achieved, efficiency directly improves 96 times, meets the high-throughout operation of biology and requires so that communicable disease can obtain quickly timely and effectively tackling when breaking out；
2nd, full-automatic, the simple to operate, used time is short, and whole flow of extracting only has four steps, especially animal tissue, cracks and centrifuges without hand-ground so that whole extraction process can substantially be completed at 30-60 minutes；
The toxic solvents such as benzene, chloroform in the 3rd, safety non-toxic, inapplicable conventional method, the injury to experiment operator is few, meets modern people-oriented, the theory of better safe than sorry；
4th, the specific binding of magnetic bead and nucleic acid make it that the nucleic acid purity height of extraction, concentration are big；
5th, using intelligent software operating system, polluted between technical ability control hole, the pollution between batch is avoided that again；Ensure the stability and repeatability of experimental result simultaneously, it is to avoid difference and mistake caused by artificial operation；
6th, reagent, instrument, consumptive material integration, for different sample materials, shoot the arrow at the target, optimize extraction conditions, readily available high-purity, high-quality nucleic acid.
Brief description of the drawings
Fig. 1 is the 96 passage Full automatic instrument for extracting nucleic acid front elevations that hatch door is opened.
Fig. 2 is that mouse liver does not grind directly extraction DNA electrophoresis results.
Fig. 3 is that plant sample DNA extracts electrophoresis result.
Fig. 4 is that mouse liver does not grind directly extraction RNA electrophoresis results.
Fig. 5 is that fin sample DNA extracts electrophoresis result.
Fig. 6 is that fecal specimens DNA extracts electrophoresis result.
Fig. 7 is that mink tissue DNA extracts electrophoresis result.
Fig. 8 is that silicon dielectric posts method extracts nucleic acid principle schematic.
Fig. 9 is method for extracting nucleic acid contrast table.
Embodiment
In order to be more readily understood the technical scheme that the present invention is provided, hereafter by the preferred embodiment of the present invention, it is described below in detail：
Embodiment 1：
1st, mouse liver DNA is extracted
Manual reagent adding operating procedure (if the kit sealed, 125 μ l lysate TR being separately added into each hole of the 1st 96 deep-well plates, add mouse liver and get over 20-60mg, then 20 μ l Proteinase Ks, 4 μ l RNase directly operate the computer)：
1) plus 250 μ l magnetic bead combination liquid CB, 125 μ l lysates TL and 125 μ l lysates TR, 20 μ l Proteinase Ks, 4 μ l RNase and about 10mg tissue samples are to the 1st 96 deep-well plates；
2) 300 μ l buffer Bs B and 20 μ l magnetic beads are added per hole in the 2nd deep-well plates；
3) 900 μ l mortifier removers IR are added per hole the 3rd, in 4 deep-well plates；
4) 900 μ l rinsing liquids WB are added per hole in the 5th deep-well plates；
5) 150 μ l elution buffers TE are added per hole in the 6th deep-well plates；
6) deep-well plates are individually positioned in the positioning seat of 96 instrument for extracting nucleic acid in order, then by stirring set insertion neck；
7) program of instrument for extracting nucleic acid is set, and specific procedure is as follows
The μ l of nucleic acid 5 after extracting are taken, agarose gel electrophoresis result is as shown in Figure 2.
Other sample extraction results are shown in three-accompanying drawing of Figure of description seven
The foregoing is only a preferred embodiment of the present invention, is not intended to limit the scope of the present invention.In every equivalent changes and modifications done according to present invention, the scope of the claims for being encompassed by the present invention.
Claims (8)
1. a kind of method of nucleic acid extraction, it is characterised in that the method for extracting nucleic acid, tissue is not required to grinding, nucleic acid is directly extracted.
2. according to the method described in claim 1, it is characterised in that reagent, instrument, consumptive material integration.
3. it is according to claim 1 tissue be not required to grinding, it is characterised in that it is described be not required to grinding refer to tissue samples do not need hand-ground, cracking, centrifuge, tissue samples are directly operated the computer.
4. reagent according to claim 2, it is characterised in that the reagent is according to different samples, using the formula of different lysates.
5. lysate according to claim 4, it is characterised in that the lysate changes solvent burden ratio on the basis of basic agent, and adds other auxiliary reagents, realizes optimization for extracting condition.
6. basic agent according to claim 5, it is characterised in that the basic agent includes guanidine hydrochloride, guanidinium isothiocyanate, Tris, Qula and led to.
7. auxiliary reagent according to claim 5, it is characterised in that the auxiliary reagent includes SLS, SDS, urea, ethanol, CTAB, PVPP etc..
8. instrument according to claim 2, it is characterised in that the instrument is instrument for extracting nucleic acid, according to different tissue samples, sets different extraction patterns.
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Cited By (4)
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| CN109632733A (en) * | 2018-11-30 | 2019-04-16 | 张丽英 | A method of utilizing bacillus cereus in ATP bioluminescence reaction detection food |
| CN109932359A (en) * | 2019-02-11 | 2019-06-25 | 张丽英 | A method of utilizing clostridium botulinum in ATP bioluminescence reaction detection food |
| CN110129312A (en) * | 2019-04-03 | 2019-08-16 | 深圳市芯思微生物科技有限公司 | Lysate and nucleic acid extraction kit |
| CN114085830A (en) * | 2022-01-12 | 2022-02-25 | 济凡生物科技（北京）有限公司 | Method for extracting microbial genome DNA (deoxyribonucleic acid) in complex sample |
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| CN103881894A (en) * | 2013-11-01 | 2014-06-25 | 艾康生物技术(杭州)有限公司 | Nucleic acid extractor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109632733A (en) * | 2018-11-30 | 2019-04-16 | 张丽英 | A method of utilizing bacillus cereus in ATP bioluminescence reaction detection food |
| CN109932359A (en) * | 2019-02-11 | 2019-06-25 | 张丽英 | A method of utilizing clostridium botulinum in ATP bioluminescence reaction detection food |
| CN110129312A (en) * | 2019-04-03 | 2019-08-16 | 深圳市芯思微生物科技有限公司 | Lysate and nucleic acid extraction kit |
| CN114085830A (en) * | 2022-01-12 | 2022-02-25 | 济凡生物科技（北京）有限公司 | Method for extracting microbial genome DNA (deoxyribonucleic acid) in complex sample |
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