CN109234271A - A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit - Google Patents
A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit Download PDFInfo
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- CN109234271A CN109234271A CN201811225692.4A CN201811225692A CN109234271A CN 109234271 A CN109234271 A CN 109234271A CN 201811225692 A CN201811225692 A CN 201811225692A CN 109234271 A CN109234271 A CN 109234271A
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- China
- Prior art keywords
- buccal swab
- genome dna
- paramagnetic particle
- reagent kit
- swab
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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 paramagnetic particle method buccal swab genome DNA extracting reagent kits, belong to extracting genome DNA field, the paramagnetic particle method buccal swab genome DNA extracting reagent kit includes: lysate and magnetic bead, lysate constituent are as follows: the guanidine hydrochloride of 20-100mmol/L, the Tris buffer solution that the PH of 10-50mmol/L is 8.0, the KCl solution of 50-250mmol/L, the EDTA chelating agent and volume content that the PH of 10-100mmol/L is 8.0 are the hydrophilic based surfactants of 0.1%-1%SDS, the paramagnetic particle method buccal swab genome DNA extracting reagent kit solves in current technology using phenol chloroform, the methods of column formulation extracts DNA process from buccal swab, and there are phenol chloroforms Pollution, the problem of DNA yield is low, purity difference, the paramagnetic particle method buccal swab genome DNA extracting reagent kit extracts the genomic DNA in buccal swab sample, and complete fragment degree is high, production concentration and purity is high, the DNA of extraction can be used for the experiment such as PCR amplification and quantitative fluorescent PCR, be worthy to be popularized.
Description
Technical field
The present invention relates to extracting genome DNA field, specifically a kind of paramagnetic particle method buccal swab extracting genome DNA reagent
Box.
Background technique
Human mouth mucous membrane is made of multi-layer cellular, has the characteristics that metabolism is fast, easy to fall off, DNA is complete, therefore, with mouth
Chamber swab acquisition port mouth desquamated cells DNA process is simple, painless and noninvasive.This method is suitable for the DNA of different age group crowd
Sample collection, the crowd for being also suitable inconvenient blood sampling acquire DNA.
The methods of phenol chloroform, Chelex-100, column formulation is mostly used to extract DNA from buccal swab in conventional method, it should
Process is there are phenol contamination with chloroform, DNA yield is low, purity difference or cumbersome, the disadvantages of with duration, has seriously affected medical care people
Member understands the efficiency of patient's patient's condition.
For the problems mentioned above in the background art, the present invention is intended to provide a kind of paramagnetic particle method buccal swab genome
DNA extraction kit.
Summary of the invention
It is above-mentioned to solve the purpose of the present invention is to provide a kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit
In the current conventional method proposed in background technique using the methods of phenol chloroform, Chelex-100, column formulation from buccal swab
Extract DNA process there are phenol contamination with chloroform, DNA yield is low, purity difference or cumbersome, the problem of with duration.
To achieve the above object, the invention provides the following technical scheme:
A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit comprising: efficient lysate and unique knot
Close magnetic bead, in which: lysate constituent are as follows: the Tris that the guanidine hydrochloride of 20-100mmol/L, the PH of 10-50mmol/L are 8.0
Buffer solution, the KCl solution of 50-250mmol/L, 10-100mmol/L PH be 8.0 EDTA chelating agent and volume content
For the hydrophilic based surfactants of 0.1%-1%SDS.
The paramagnetic particle method buccal swab genome DNA extracting reagent kit application method is the following steps are included: buccal swab takes
It cell and cellular liquid will be contained is transferred to centrifuge tube, protease K digesting, magnetic beads for purifying, 75% on sample, lysate elution swab
Ethanol solution washing and the elution of the water low salt buffer of free containing Nuclease.
As a further solution of the present invention: the buccal swab takes sample embodiment are as follows: will wipe across in oral cavity
Swab perhaps swab is cut in the centrifuge tube of 1.5mL or it is made to fall off by cotton swab transposition；The lysate elutes swab
Upper cell simultaneously will contain cellular liquid and be transferred to centrifuge tube embodiment are as follows: be added into equipped with the centrifuge tube in mouth swab sample
300uL lysate, concussion mix centrifugation, liquid are transferred in new 1.5mL centrifuge tube and obtain mixed liquor one.
As further scheme of the invention: the protease K digesting embodiment are as follows: be added into mixed liquor one
10uL Proteinase K, piping and druming mix, and 30min is incubated under conditions of 56 DEG C, the revolving speed of 800rpm and obtains mixed liquor two；The magnetic bead
Purify embodiment are as follows: after mixed liquor two is placed at room temperature for 5min, 12000rpm is centrifuged 2min, and 200uL supernatant is transferred to and is contained
It in the 1.5mL centrifuge tube of 100uL combination magnetic bead, is vortexed and mixes, be stored at room temperature and be incubated for 5min, it is short from centrifuge tube is placed in magnetic force
Frame is clarified up to solution.
As the further scheme of the present invention: 75% ethanol solution washs embodiment are as follows: remove supernatant, to from
It is added 500uL75% ethanol solution in heart pipe, violent Vortex, 30s on magnetic frame, it is primary to repeat the step, i.e. 75% ethyl alcohol
Solution washing step implement twice, it is short from, using pipettor remove bottom residual ethanol solution, 2-5min is stored at room temperature, until magnetic
Pearl is non-reflective；The water of free containing the Nuclease low salt buffer type of elution are as follows: the less salt buffering of 20-50uL is added
Liquid is removed centrifuge tube from magnetic frame, and magnetic bead is resuspended, is stored at room temperature 5min, centrifuge tube is placed in 2min on magnetic frame, uses liquid relief
Device Aspirate supernatant is transferred in new 1.5ml centrifuge tube.
Compared with prior art, the beneficial effects of the present invention are:
Rationally, sample (buccal swab) is logical for a kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit design
It crosses and cracks digestion under the action of efficient lysate and Proteinase K, DNA is discharged into lysate, unique magnetic bead is added
(having contained specific reaction system) specifically binds it with DNA.In a certain proportion of ethanol solution, by the egg in the system
White matter and remaining impurity remove, and leave behind the DNA combined on magnetic bead, last DNA is washed in aqueous solution or low salt buffer
De-, obtained DNA can be used for subsequent experimental.
A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit is practical, and it is suitable for extracting oral cavity
The genomic DNA of swab sample, A260/280 are greater than 1.8, and complete fragment degree is high；The whole process of extraction is not related to toxic having
Evil reagent, safe operation process, whole process only need the extraction that high quality DNA can be completed in 60 minutes, facilitate convenient and quick stabilization；It is high
The cracking liquid system of effect and unique magnetic bead match, and the operation used time is short, and the DNA of production concentration and purity is high, extraction can be used for
The experiment such as high-flux sequence experiment, digestion, PCR amplification, quantitative fluorescent PCR.
A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit is worth the genome in buccal swab sample
DNA extracts field and promotes and use.
Detailed description of the invention
Fig. 1 is a kind of application method flow chart of paramagnetic particle method buccal swab genome DNA extracting reagent kit.
Specific embodiment
Technical solution of the present invention is described in more detail With reference to embodiment.
Embodiment
Referring to Fig. 1, a kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit comprising: efficient lysate with
And unique combination magnetic bead, wherein lysate constituent are as follows: the guanidine hydrochloride of 20-100mmol/L, the PH of 10-50mmol/L are
8.0 Tris buffer solution, the KCl solution of 50-250mmol/L, 10-100mmol/L PH be 8.0 EDTA chelating agent and
Volume content is the hydrophilic based surfactants of 0.1%-1%SDS.
The paramagnetic particle method buccal swab genome DNA extracting reagent kit application method is the following steps are included: buccal swab takes
It cell and cellular liquid will be contained is transferred to centrifuge tube, protease K digesting, magnetic beads for purifying, 75% on sample, lysate elution swab
Ethanol solution washing and the elution of Nuclease free water/ low salt buffer.
The buccal swab takes sample embodiment are as follows: by the swab wiped across in oral cavity or cotton swab transposition in
In the centrifuge tube of 1.5mL, swab is cut or it is made to fall off.
It cell and cellular liquid will be contained is transferred to centrifuge tube embodiment on lysate elution swab are as follows: toward being equipped with mouth
300uL lysate is added in centrifuge tube in chamber swab samples, concussion mixes centrifugation, and liquid is transferred to new 1.5mL and is centrifuged
(about 200mL) obtains mixed liquor one in pipe.
The protease K digesting embodiment are as follows: 10uL Proteinase K is added into mixed liquor one, piping and druming mixes, 56
DEG C, it is incubated for 30min under conditions of the revolving speed of 800rpm and obtains mixed liquor two.
The magnetic beads for purifying embodiment are as follows: after mixed liquor two is placed at room temperature for 5min, 12000rpm is centrifuged 2min, will
200uL supernatant is transferred in the 1.5mL centrifuge tube of the magnetic bead of combination containing 100uL, be vortexed mix, be stored at room temperature be incubated for 5min, it is short from,
Centrifuge tube is placed in magnetic frame to clarify up to solution.
75% ethanol solution washs embodiment are as follows: removes supernatant, 75% ethyl alcohol of 500uL is added into centrifuge tube
Solution, violent Vortex, 30s on magnetic frame repeat that the step is primary, i.e. 75% ethanol solution washing step is implemented twice, short
From, using pipettor remove bottom residual ethanol solution, 2-5min is stored at room temperature, until magnetic bead is non-reflective.
The water of free containing the Nuclease low salt buffer type of elution are as follows: 20-50uL is added contains Nuclease
Free water low salt buffer is removed centrifuge tube from magnetic frame, and magnetic bead is resuspended, is stored at room temperature 5min, centrifuge tube is placed in
2min on magnetic frame is transferred in new 1.5ml centrifuge tube with pipettor Aspirate supernatant.
Comparative example
The column formulation of commodity in use extracts buccal swab genomic DNA, extracts oral cavity with the paramagnetic particle method that embodiment provides
Swab genomic DNA method is compared, and the column formulation extraction buccal swab genomic DNA operation of the commercialization is relative complex, and
It is not easy to automatic operation, and the buccal swab genomic DNA concentration extracted is inadequate, purity is lower.
The operation principle of the present invention is that:
Sample (buccal swab) is digested by cracking under the action of efficient lysate and Proteinase K, and DNA is discharged into
In lysate, unique magnetic bead (having contained specific reaction system), which is added, specifically binds it with DNA.In a certain proportion of second
In alcoholic solution, the protein in the system is removed with remaining impurity, leaves behind the DNA combined on magnetic bead, last DNA is water-soluble
It is eluted in liquid or low salt buffer, obtained DNA can be used for subsequent experimental.
A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit design rationally, extracts buccal swab sample
This genomic DNA, A260/280 are greater than 1.8, and complete fragment degree is high, and the whole process of extraction is not related to poisonous and harmful examination
Agent, safe operation process, whole process only need the extraction that high quality DNA can be completed in 60 minutes, facilitate convenient and quick stabilization, Er Qiecao
Short when effect, production concentration and purity is high, the DNA of extraction can be used for high-flux sequence experiment, digestion, PCR amplification, fluorescent quantitation
The experiment such as PCR, a kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit are worth the gene in buccal swab sample
Group DNA extracts field and promotes and use.
Better embodiment of the invention is explained in detail above, but the present invention is not limited to above-mentioned embodiment party
Formula within the knowledge of one of ordinary skill in the art can also be without departing from the purpose of the present invention
Various changes can be made.
Claims (7)
1. a kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit comprising: lysate and magnetic bead, it is characterised in that:
The lysate constituent are as follows: the guanidine hydrochloride of 20-100mmol/L, 10-50mmol/L PH be 8.0 Tris buffer solution,
The EDTA chelating agent and volume content that the KCl solution of 50-250mmol/L, the PH of 10-100mmol/L are 8.0 are 0.1%-
The hydrophilic based surfactants of 1%SDS.
2. a kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit according to claim 1, it is characterised in that: institute
State lysate constituent are as follows: the guanidine hydrochloride of 30-90mmol/L, 20-40mmol/L PH be 8.0 Tris buffer solution, 60-
The EDTA chelating agent and volume content that the KCl solution of 240mmol/L, the PH of 20-90mmol/L are 8.0 are 0.2%-0.9%
The hydrophilic based surfactants of SDS.
3. a kind of application method of the paramagnetic particle method buccal swab genome DNA extracting reagent kit as described in claim 1-2 is any,
Characterized by the following steps: buccal swab takes sample, lysate to elute cell on swab and will contain cellular liquid transfer
To the less salt of centrifuge tube, protease K digesting, magnetic beads for purifying, the washing of 75% ethanol solution and the water of free containing Nuclease
Buffer elution.
4. a kind of application method of paramagnetic particle method buccal swab genome DNA extracting reagent kit according to claim 3, special
Sign is: the buccal swab takes sample embodiment are as follows: by the swab wiped across in oral cavity or cotton swab transposition in 1.5mL
Centrifuge tube in, swab is cut or it is made to fall off；The lysate elutes cell on swab and will contain cellular liquid transfer
To centrifuge tube embodiment are as follows: be added 300uL lysate into equipped with the centrifuge tube in mouth swab sample, concussion mix from
Liquid is transferred in new 1.5mL centrifuge tube and obtains mixed liquor one by the heart.
5. a kind of application method of paramagnetic particle method buccal swab genome DNA extracting reagent kit according to claim 4, special
Sign is: the protease K digesting embodiment are as follows: 10uL Proteinase K is added into mixed liquor one, piping and druming mixes, and at 56 DEG C,
It is incubated for 30min under conditions of the revolving speed of 800rpm and obtains mixed liquor two；The magnetic beads for purifying embodiment are as follows: two room temperature of mixed liquor is put
After setting 5min, it is centrifuged 2min under the conditions of 12000rpm centrifugal rotational speed, 200uL supernatant is transferred to the magnetic bead of combination containing 100uL
In 1.5mL centrifuge tube, be vortexed mix, be stored at room temperature be incubated for 5min, it is short from, by centrifuge tube be placed in magnetic frame up to solution clarify.
6. a kind of application method of paramagnetic particle method buccal swab genome DNA extracting reagent kit according to claim 5, special
Sign is: 75% ethanol solution washs embodiment are as follows: removes supernatant, 75% ethyl alcohol of 500uL is added into centrifuge tube
Solution, violent Vortex, 30s on magnetic frame repeat that the step is primary, i.e. 75% ethanol solution washing step is implemented twice, short
From, using pipettor remove bottom residual ethanol solution, 2-5min is stored at room temperature, until magnetic bead is non-reflective.
7. a kind of application method of paramagnetic particle method buccal swab genome DNA extracting reagent kit according to claim 6, special
Sign is: the water low salt buffer of free containing Nuclease elutes embodiment are as follows: containing for 20-50uL is added
Nuclease free water low salt buffer, removes centrifuge tube from magnetic frame, and magnetic bead is resuspended, is stored at room temperature 5min, will be from
Heart pipe is placed in 2min on magnetic frame, with pipettor Aspirate supernatant, is transferred in new 1.5ml centrifuge tube.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811225692.4A CN109234271A (en) | 2018-10-21 | 2018-10-21 | A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit |
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| CN201811225692.4A CN109234271A (en) | 2018-10-21 | 2018-10-21 | A kind of paramagnetic particle method buccal swab genome DNA extracting reagent kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110273027A (en) * | 2019-06-20 | 2019-09-24 | 上海伯杰医疗科技有限公司 | Norovirus G I, G II and IV type nucleic acid parting detecting reagent of G and detection method |
| CN111778240A (en) * | 2020-07-23 | 2020-10-16 | 北京果壳生物科技有限公司 | Method for rapidly extracting genome DNA (deoxyribonucleic acid) from various samples based on paramagnetic particle method |
| CN114164207A (en) * | 2022-02-11 | 2022-03-11 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
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| CN106350509A (en) * | 2016-10-18 | 2017-01-25 | 哈尔滨博泰生物科技有限公司 | Fast and efficient saliva DNA extraction kit and extraction method |
| CN107058286A (en) * | 2016-12-29 | 2017-08-18 | 苏州英芮诚生化科技有限公司 | The kit and its application method of oral cavity sample genomic dna are extracted based on paramagnetic particle method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN106350509A (en) * | 2016-10-18 | 2017-01-25 | 哈尔滨博泰生物科技有限公司 | Fast and efficient saliva DNA extraction kit and extraction method |
| CN107058286A (en) * | 2016-12-29 | 2017-08-18 | 苏州英芮诚生化科技有限公司 | The kit and its application method of oral cavity sample genomic dna are extracted based on paramagnetic particle method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110273027A (en) * | 2019-06-20 | 2019-09-24 | 上海伯杰医疗科技有限公司 | Norovirus G I, G II and IV type nucleic acid parting detecting reagent of G and detection method |
| CN110273027B (en) * | 2019-06-20 | 2021-09-07 | 上海伯杰医疗科技有限公司 | Nucleic acid typing detection kit and detection method for norovirus GII, GII and GIV |
| CN111778240A (en) * | 2020-07-23 | 2020-10-16 | 北京果壳生物科技有限公司 | Method for rapidly extracting genome DNA (deoxyribonucleic acid) from various samples based on paramagnetic particle method |
| CN114164207A (en) * | 2022-02-11 | 2022-03-11 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
| CN114164207B (en) * | 2022-02-11 | 2022-05-17 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
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