CN106834277A - A kind of paramagnetic particle method separates the method and separating kit of dissociative DNA - Google Patents
A kind of paramagnetic particle method separates the method and separating kit of dissociative DNA Download PDFInfo
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- CN106834277A CN106834277A CN201710138280.6A CN201710138280A CN106834277A CN 106834277 A CN106834277 A CN 106834277A CN 201710138280 A CN201710138280 A CN 201710138280A CN 106834277 A CN106834277 A CN 106834277A
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- concentration
- guanidinesalt
- alcohols
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- nucleic acid
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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
The invention discloses a kind of method that dissociative DNA is separated with paramagnetic particle method, it is characterised in that extracting method step of the invention includes that S1 discharges free nucleic acid and is adsorbed in magnetic bead surfaces under given conditions；The impurity such as S2 washing removal chemical substance and protein；S3 affords DNA, dissociative DNA extracting method provided by the present invention and kit have that easy, quick, yield is high, product purity is high, the advantage such as cheap, suitable for the quick separating DNA from the acellular body fluid such as serum, blood plasma, gained DNA may be directly applied to PCR equimolecular biological experiment.
Description
Technical field
Method and the separation of dissociative DNA are separated from the acellular body fluid such as serum, blood plasma the present invention relates to a kind of use magnetic bead
Kit.
Background technology
Free nucleic acid is earliest the forties in 20th century two French scientists Mandel and Metais discovery earliest.They
Research be published in nineteen forty-sevenOn DE BIOLOGIE DE STRASBOURG magazines.1997, YMD Lo etc. existed
Internationally famous medical journals《Lancet》(Lancet) on, it was recently reported that Y dyes are detected in the peripheral blood for nourishing male tire pregnant woman
Colour solid is present, the dissociative DNA that there is fetus in maternal blood so as to demonstrate.Then, correlative study finds fetus dissociative again
Detection times of the DNA in maternal blood, mainly relevant with gestational age, gestational age is more early, and fetus composition is relatively fewer.This discovery
Noninvasive antenatal detection is carried out to pregnant woman has important diagnostic significance.This provides a kind of effective approach to non-invasive diagnosis.But
In blood plasma the DNA of maternal source account for STb gene ratio it is very high, improve to a certain extent detection foetal DNA difficulty.Mesh
Before, dissociative DNA is clinically mainly used in the property DNA detections of father source, such as sry gene type identification sex of foetus, detection RhD blood groups
And other father source property genetic disease detections.Pre-natal diagnosis is most commonly that the aneuploid disease detection such as trisomy 21, and it is required
Accurate quantification is carried out to chromosome copies quantity, this is a challenge for dissociative DNA.
At present, extracting the method for the commercialization of nucleic acid mainly has silicon fiml that column adsorption method and magnetic bead absorption method is centrifuged.Silicon fiml from
Stem adsorption method principle is the other materials such as impurity such as chemicals, carbohydrate, albumen by nucleic acid absorption to silica surface
Quickly washed away with cleaning solution, then use elution nucleic acid, the method is simple and quick, can effectively removed various in sample
Impurity, obtains the DNA higher compared with purity, but relatively costly, it is necessary to multistep high speed centrifugation, changes centrifuge tube, not high-throughout
Instrument is supporting, is not suitable for clinical extensive pattern detection.It is the nucleic acid extraction for just growing up in recent years that paramagnetic particle method extracts nucleic acid
Method.Its cardinal principle is to introduce active group by combined polymerization and surface modification etc. on magnetic bead (predominantly ferrite) surface,
These active groups have hydroxyl, carboxyl, amino, sulfydryl etc., are prepared into the magnetic affinity adsorbent of tool, then common with nucleic acid
After incubation, by principles such as hydrophobic effect, electrostatic interaction, ionic adsorptions, make nucleic acid absorption to magnetic bead surfaces, by simply washing
Wash, Magneto separate, obtain to surface and be combined with the bead complexes of nucleic acid, changing the conditions such as reagent pH value can be nucleic acid from magnetic bead
Elute.The method is easy to operate, quick, and the nucleic acid purity of acquisition is high, and yield is big, and existing many companies develop and match somebody with somebody at present
The automated system of set, can effectively improve operating efficiency.
At present, the paramagnetic particle method free nucleic acid extracts kit complex operation of commercialization is, it is necessary to using Proteinase K in 37-56
DEG C sample 10-30 minute is incubated, absolute ethyl alcohol or isopropanol are added after being subsequently cooled to room temperature, addition bead suspension is mixed
It is even, 5-10 minutes is stood, dried 10-15 minutes by being placed in room temperature after a series of rinse steps.The triviality of step causes it
Automation mechanized operation low degree, instrument universality is poor.Therefore in the urgent need to developing the free core of paramagnetic particle method easy to operate, stability and high efficiency
Sour extracts kit.
The content of the invention
The present patent application content is related to biomedical sector, intends application one kind and divides from the acellular body fluid such as serum, blood plasma
From the method and extracts reagent of dissociative DNA.
Extracting method step of the invention includes：1) discharge free nucleic acid and be adsorbed in magnetic bead surfaces under given conditions；
2) impurity such as washing removal chemical substance and protein；3) DNA is afforded.
The dissociative DNA extracting method that the present invention is provided, it is characterised in that：
1) release nucleic acid cracking used combines the guanidinesalt containing high concentration in liquid, in being guanidine hydrochloride, guanidinium isothiocyanate
One or two, the concentration of guanidinesalt is 1-6M.
2) release nucleic acid cracking used combines the surfactant containing 1-20% in liquid, and the surfactant can be
One or more in Triton X-100, Tween 20, SDS, SLS, NP-40；
3) release nucleic acid cracking used combines the alcohols containing high concentration in liquid, can be ethanol, isopropanol, polyethylene glycol
One or more, the concentration of alcohol is 10%-100%；
4) adsorbing medium is the magnetic bead of surface modification, and modification group is hydroxyl, carboxyl, one or two in amino；
5) alcohols containing high concentration in cleaning solution, can be one or two in ethanol, isopropanol；The concentration of alcohol is
50%-100%
6) salt containing high concentration in cleaning solution, in being guanidine hydrochloride, guanidinium isothiocyanate, lithium chloride, sodium citrate
One or more；Concentration is 0.5-5M
7) eluent used can be water when eluting, and can also be the water containing low concentration of salt ion.The concentration of salt is
1mM-100mM
The extracts kit that the present invention is provided, including following several components：Cracking with reference to liquid, cleaning solution, eluent I, wash
Wash liquid II, magnetic bead, operation instructions.
The extracts kit that the present invention is provided, it is characterised in that：
1) release nucleic acid cracking used combines the guanidinesalt containing high concentration in liquid, in being guanidine hydrochloride, guanidinium isothiocyanate
One or two, the concentration of guanidinesalt is 1-6M.
2) release nucleic acid cracking used combines the surfactant containing 1-20% in liquid, and the surfactant can be
One or more in Triton X-100, Tween 20, SDS, SLS, NP-40；
3) release nucleic acid cracking used combines the alcohols containing high concentration in liquid, can be ethanol, isopropanol, polyethylene glycol
One or more, the concentration of alcohol is 10%-100%；
4) adsorbing medium is the magnetic bead of surface modification, and modification group is hydroxyl, carboxyl, one or two in amino；
5) alcohols containing high concentration in cleaning solution, can be one or two in ethanol, isopropanol；The concentration of alcohol is
50%-100%
6) salt containing high concentration in cleaning solution, in being guanidine hydrochloride, guanidinium isothiocyanate, lithium chloride, sodium citrate
One or more；Concentration is 0.5-5M
7) eluent used can be water when eluting, and can also be the water containing low concentration of salt ion.The concentration of salt is
1mM-100mM
Dissociative DNA extracting method provided by the present invention and kit have that easy, quick, yield is high, product purity is high,
The advantage such as cheap, it is adaptable to which the quick separating DNA from the acellular body fluid such as serum, blood plasma, gained DNA may be directly applied to gather
Polymerase chain reacts equimolecular biological experiment.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be to that will use needed for embodiment description
Accompanying drawing be briefly described, it should be apparent that, drawings in the following description are some embodiments of the present invention, for this area
For those of ordinary skill, on the premise of not paying creative work, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the flow chart of the embodiment of the present invention；
Fig. 2 is the operating procedure schematic diagram of the embodiment of the present invention.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is a part of embodiment of the invention, rather than whole embodiments.Based on this hair
Embodiment in bright, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example, belongs to the scope of protection of the invention.
Although present disclosure is illustrated with reference to this example, the limit to patent of the present invention is not construed as
System, it is intended that the scope of the present invention be defined by the claims appended hereto.In addition, those skilled in the art limits in appended claims
In the range of various changes or modification are carried out to the present invention, these change or modified forms equally belong to guarantor of the invention
Shield scope.
The agent formulations of embodiment 1
Cracking combines liquid：2.8M GuSCN, 50mM Tris (pH 7.5), 5%Triton X-100,30%IPA；
Cleaning solution I：5M LiCl, 40% ethanol
Cleaning solution II：80% ethanol
Eluent：5mM Tris (pH 8.0), 0.5EDTA, DEPC water
Embodiment 2 is dissociated RNA extraction method
1) 1.5mL centrifuge tubes are taken, the blood plasma or serum of 200 μ L fresh separateds is added, adds 600 μ L cracking to combine liquid, plus
Enter the vibration of 1mg magnetic beads to mix.
2) room temperature places 10min.
3) Magneto separate, sucks supernatant.
4) 800 μ L cleaning solution I are added, is vortexed and is mixed.
5) Magneto separate, sucks supernatant.
6) 800 μ L cleaning solution IIs are added, is vortexed and is mixed.
7) Magneto separate, sucks supernatant.
8) uncap, drying at room temperature 5min.
9) 50 μ L eluents are added, is vortexed and is mixed.
10) Magneto separate, collects supernatant, -20 DEG C or -80 DEG C preservations.
In the above-described embodiments, the description to each embodiment all emphasizes particularly on different fields, and is not described in certain embodiment
Part, may refer to the associated description of other embodiment.
The content download method and relevant device that are there is provided the embodiment of the present invention above, system are described in detail,
Specific case used herein is set forth to principle of the invention and implementation method, and the explanation of above example is use
Understand the method for the present invention and its core concept in help；Simultaneously for those of ordinary skill in the art, according to of the invention
Thought, will change in specific embodiments and applications, and in sum, this specification content should not be construed as
Limitation of the present invention.
Claims (10)
1. a kind of method of use Beads enrichment dissociative DNA, it is characterised in that methods described includes：
S1 release free nucleic acid simultaneously free nucleic acid is adsorbed in the surface of the magnetic bead, wherein the magnetic bead by surface modification at
Reason, the modification group for using is for one or two in hydroxyl, carboxyl and amino；
S2 is washed, wherein the cleaning solution that the washing is used includes alcohol and compound salt, the alcohol is in ethanol, isopropanol
Plant or two kinds, the concentration of the alcohol is 50%-100%, the compound salt is guanidine hydrochloride, guanidinium isothiocyanate, lithium chloride and lemon
One or more in lemon acid sodium, the concentration of the compound salt is 0.5-5M；
S3 is eluted.
2. method according to claim 1, it is characterised in that the cracking knot that free nucleic acid is used is discharged in the step S1
Contain guanidinesalt, surfactant and alcohols in conjunction liquid, wherein
The guanidinesalt is one or two in guanidine hydrochloride and guanidinium isothiocyanate, the surfactant be Triton X-100,
One or more in Tween 20, SDS, SLS and NP-40, the alcohols be ethanol, isopropanol and polyethylene glycol one kind or
It is various.
3. method according to claim 2, it is characterised in that the concentration of the guanidinesalt in the cracking combination liquid is 1-6M.
4. method according to claim 2, it is characterised in that the concentration of the alcohols in the cracking combination liquid is 10%-
100%.
5. a kind of extracts kit of utilization Beads enrichment dissociative DNA, described to carry using method as claimed in claim 1 or 2
Taking kit includes following several components：Cracking combines liquid, cleaning solution I, eluent I, cleaning solution II and magnetic bead.
6. extracts kit according to claim 5, it is characterised in that the magnetic bead is processed by surface modification, uses
Modification group be one or two in hydroxyl, carboxyl and amino.
7. extracts kit according to claim 5, it is characterised in that contain guanidinesalt, surface in the cracking combination liquid
Activating agent and alcohols,
Wherein, the guanidinesalt is one or two in guanidine hydrochloride and guanidinium isothiocyanate, and the surfactant is Triton X-
100th, one or more in Tween 20, SDS, SLS and NP-40, the alcohols is the one of ethanol, isopropanol and polyethylene glycol
Plant or various.
8. extracts kit according to claim 5, it is characterised in that the concentration of the guanidinesalt in the cracking combination liquid is
1-6M。
9. extracts kit according to claim 5, it is characterised in that the concentration of the alcohols in the cracking combination liquid is
10%-100%.
10. extracts kit according to claim 5, it is characterised in that the cleaning solution I includes LiCl.
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| CN2017100746966 | 2017-02-11 | ||
| CN201710074696 | 2017-02-11 |
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Cited By (11)
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| CN107254463A (en) * | 2017-06-20 | 2017-10-17 | 迈克生物股份有限公司 | Kit for extracting HCV RNA |
| CN107723285A (en) * | 2017-10-18 | 2018-02-23 | 中山大学肿瘤防治中心 | A kind of Total DNA extraction method and extracts kit for being common to standard biologic sample |
| CN108220284A (en) * | 2018-01-23 | 2018-06-29 | 南昌大学 | A kind of paramagnetic particle method bacterial genomes DNA extraction kit and application |
| CN108220282A (en) * | 2017-12-22 | 2018-06-29 | 德诺杰亿（北京）生物科技有限公司 | The extracts reagent and extracting method of a kind of dissociative DNA |
| CN108949747A (en) * | 2018-07-27 | 2018-12-07 | 广州奇辉生物科技有限公司 | A kind of kit and application method extracting RNA |
| CN109022420A (en) * | 2018-08-29 | 2018-12-18 | 武汉纳磁生物科技有限公司 | A kind of DNA extraction method based on magnetic bead, lysate and kit |
| CN109371010A (en) * | 2018-11-26 | 2019-02-22 | 广东腾飞基因科技股份有限公司 | A kind of paramagnetic particle method plasma DNA extracts kit and extracting method |
| CN110144345A (en) * | 2019-05-10 | 2019-08-20 | 上海交通大学 | A method of extracting cfDNA from liquor folliculi |
| CN110951725A (en) * | 2019-12-30 | 2020-04-03 | 申友基因组研究院(南京)有限公司 | One-step nucleic acid extraction process based on paramagnetic particle method |
| CN111235314A (en) * | 2020-03-13 | 2020-06-05 | 苏州白垩纪生物科技有限公司 | Virus inactivation, capture and real-time fluorescence isothermal amplification detection kit and application thereof |
| CN113373141A (en) * | 2021-07-23 | 2021-09-10 | 中国科学院宁波材料技术与工程研究所 | Extraction method of free DNA |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254463A (en) * | 2017-06-20 | 2017-10-17 | 迈克生物股份有限公司 | Kit for extracting HCV RNA |
| CN107254463B (en) * | 2017-06-20 | 2018-11-20 | 迈克生物股份有限公司 | For extracting the kit of HCV RNA |
| CN107723285A (en) * | 2017-10-18 | 2018-02-23 | 中山大学肿瘤防治中心 | A kind of Total DNA extraction method and extracts kit for being common to standard biologic sample |
| CN108220282A (en) * | 2017-12-22 | 2018-06-29 | 德诺杰亿（北京）生物科技有限公司 | The extracts reagent and extracting method of a kind of dissociative DNA |
| CN108220284A (en) * | 2018-01-23 | 2018-06-29 | 南昌大学 | A kind of paramagnetic particle method bacterial genomes DNA extraction kit and application |
| CN108949747A (en) * | 2018-07-27 | 2018-12-07 | 广州奇辉生物科技有限公司 | A kind of kit and application method extracting RNA |
| CN109022420A (en) * | 2018-08-29 | 2018-12-18 | 武汉纳磁生物科技有限公司 | A kind of DNA extraction method based on magnetic bead, lysate and kit |
| CN109371010A (en) * | 2018-11-26 | 2019-02-22 | 广东腾飞基因科技股份有限公司 | A kind of paramagnetic particle method plasma DNA extracts kit and extracting method |
| CN110144345A (en) * | 2019-05-10 | 2019-08-20 | 上海交通大学 | A method of extracting cfDNA from liquor folliculi |
| CN110144345B (en) * | 2019-05-10 | 2022-07-15 | 上海交通大学 | Method for extracting cfDNA from follicular fluid |
| CN110951725A (en) * | 2019-12-30 | 2020-04-03 | 申友基因组研究院(南京)有限公司 | One-step nucleic acid extraction process based on paramagnetic particle method |
| CN111235314A (en) * | 2020-03-13 | 2020-06-05 | 苏州白垩纪生物科技有限公司 | Virus inactivation, capture and real-time fluorescence isothermal amplification detection kit and application thereof |
| CN113373141A (en) * | 2021-07-23 | 2021-09-10 | 中国科学院宁波材料技术与工程研究所 | Extraction method of free DNA |
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Application publication date: 20170613 |