CN109182331A - A kind of paramagnetic particle method extracts the kit and extracting method of DNA - Google Patents
A kind of paramagnetic particle method extracts the kit and extracting method of DNA Download PDFInfo
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- CN109182331A CN109182331A CN201811143843.1A CN201811143843A CN109182331A CN 109182331 A CN109182331 A CN 109182331A CN 201811143843 A CN201811143843 A CN 201811143843A CN 109182331 A CN109182331 A CN 109182331A
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- dna
- magnetic bead
- kit
- liquid
- paramagnetic particle
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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 patent discloses the kit and extracting method of a kind of paramagnetic particle method extraction DNA, and kit includes digestion cracking system, articulated system, rinsing system, the big system of eluent system four.Kit extracting method includes: sample homogenization, cell cracking, centrifugation, DNA in conjunction with magnetic bead, washing, DNA elution.The present invention is had an advantage that: (1) avoiding complicated Liquid-treatment processes, the whole operating time is shortened 20min or more, keeps extraction process more quick；(2) no liquid residual in magnetic bead transfer process, elution efficiency and PCR reaction have higher success rate；(3) in operating process, bar magnet set can be used for multiple times, and improve the efficiency of consumptive material, and cost reduces by 30% or more.
Description
Technical field
The present invention relates to kit and extraction sides that DNA is extracted in a kind of extraction field DNA more particularly to a kind of paramagnetic particle method
Method.
Background technique
It is more and more real since the automation sample processing apparatus of First clinical research in 1980 is put into use
It tests room automated system and provides opportunity to solve laboratory facing challenges.Due to having quick, accurate and repetition reliability
The use of strong ability to work, automated system can greatly improve laboratory work efficiency, bring powerful high-throughput sample
Processing capacity.The fast development of the demand driving laboratory automation system in market, while having driven nucleic acid extraction technology and city
Field is kept updating, and full-automatic nucleic acid extraction system will gradually replace manual operation under main trend.Currently, more foreign countries
The nucleic acid extraction system of well-known manufacturer occupies leading market status, monopolizes most domestic market, while home equipment is also being put forth energy
It rises and directly chases after, product and technology are all more mature.Entire domestic market, full-automatic minim DNA extraction system mainly by QIAGEN,
Occupied by the foreign brand names such as Hamilton and Tecan, platform is at high cost, and extraction rate is slow, existing in actual use
The full-automatic extraction platform having can not often cope with the urgent case of burst, to become the chicken ribs in laboratory.
While increasingly important function is played in forensic field, DNA extraction system also has become to be worked as DNA technique
The modern essential Experimental Establishment of forensic laboratory.Current experiment room nucleic acid extraction system mostly uses manual method or semi-automatic
Formula, very complicated not only delay the time of clear up a criminal case, also affect the detection efficiency of case, increasingly become restriction case
The bottleneck of part detection, causes demand of the front man to full automatic treatment system more and more eager.And it is at low cost, high-efficient
Full-automatic minim DNA extract workbench at trend of the times.
DNA extraction method provided by the invention includes the programs such as sample digestion cracking, centrifugation, combination, rinsing, elution, real
The automation of existing overall process, operating in a key, may be implemented inexpensive, efficient, quick DNA extraction process, be suspicion of crime
The detection of the investigation and case of people provides most effective help.
Summary of the invention
For the deficiency and disadvantage of traditional scheme, the present invention provides kit and the extraction side that a kind of paramagnetic particle method extracts DNA
Method, can overcome the defect of conventional method, to achieve the purpose that high efficiency, low cost.
A kind of paramagnetic particle method extracts the kit of DNA, including digestion cracking system, articulated system, rinsing system, eluent system
Four big systems.
The digestion cracking system includes digestion buffer, protease, dithiothreitol (DTT).
The articulated system includes magnetic bead and combines liquid, and the magnetic bead is received for the superparamagnetism of specified particle diameter and surface modification
Rice grain, the combination liquid provides low ph conditions with high salt for magnetic bead adsorption of DNA, so that DNA is effectively combined with magnetic bead surfaces hydroxyl.
The rinsing system is ethanol solution.
The eluent system includes eluent.
The method of kit extraction DNA: carrying out digestion cracking first, so that DNA separate out from cell, then be centrifuged
Except carrier and other solid impurities, magnetic bead adsorption of DNA in combining pendular ring border, magnet absorption magnetic bead simultaneously discards solution, rinsing liquid
The soluble impurity of magnetic bead surfaces is washed away, the DNA that eluent washes lower magnetic bead absorption uses the Magneto separate mode of bar magnet method, passes through spy
The bar magnet of system carries out shifting sample while transfer magnetic bead, and magnetic bead orientation is gathered on the special magnetosheath that bar magnet surface is cased with, and leads to
Cross bar magnet set movement come realize magnetic bead from sample combination liquid to rinsing liquid, then to eluent transfer, to complete nucleic acid
Automate extraction process.
The kit extracts the performance indicator of DNA method:
The DNA rate of recovery >=70%；
Using independent centrifuge, maximum centrifugal force 1000g；
The magnetic bead rate of recovery >=95%；
Single maximum sample treating capacity: 48 samples；
Separate run times≤2h；
Single sample maximum handles volume: 1300 μ l；
Minimum elution volume: 25 μ l；
Liquid relief precision CV value≤5%；
Operation sequence >=500 can be stored.
Beneficial effects of the present invention: (1) avoiding complicated Liquid-treatment processes, and the whole operating time is shortened
20min or more keeps extraction process more quick；(2) no liquid residual in magnetic bead transfer process, elution efficiency and PCR reaction
It has higher success rate；(3) in operating process, bar magnet set can be used for multiple times, and improve the efficiency of consumptive material, cost reduce by 30% with
On.
Detailed description of the invention
Fig. 1 is the embodiment of the present invention operational flowchart.
Specific preparation method
The present invention is described in detail with specific example with reference to the accompanying drawing.
As shown in Figure 1, the kit extracting method that a kind of paramagnetic particle method extracts DNA is as follows:
(1) it sample homogenization: to the type and proportion of chemical reagent in digestion buffer, and fullys shake and shakes up, guarantee body
System uniformly, stands 10 minutes under 25 degrees Celsius of room temperature after mixing.
(2) cell cracking: system is placed in 50 degrees Celsius of water-bath and is cracked 20 to 30 minutes, and system is persistently shaken
It swings, digestion buffer, protease, dithiothreitol (DTT) provide buffer condition for digestion cracking process, the DNA in digestion cracking process
With Separation of Proteins, released from nucleus.
(3) it is centrifuged: using independent centrifuge, maximum centrifugal force 1000g centrifugation time 2 to 5 minutes, is made by centrifugation
With to be separated by solid-liquid separation, carrier and impurity are removed.
(4) DNA is in conjunction with magnetic bead: magnetic bead is the superparamagnetic nano particle of specified particle diameter and surface modification, is in conjunction with liquid
Magnetic bead adsorption of DNA provides low ph conditions with high salt, so that DNA is effectively combined with magnetic bead surfaces hydroxyl, magnetic bead is in combining pendular ring border
Adsorption of DNA, the magnetic bead rate of recovery are stablized 95% or more.
(5) it washs: cleaning the soluble impurity of magnetic bead surfaces, main wash includes salt ion, protein and inhibition
Impurity, the ingredient of rinsing liquid are ethanol solutions, and washing times are 2 times.
(6) DNA is eluted: the eluent of 25 μ l is added, is uniformly mixed, elutes what magnetic bead surfaces combined under low-salt conditions
DNA can collect the DNA of purifying after separation, save under conditions of subzero 20 to subzero 80 degrees Celsius, DNA recovery efficiency is steady
It is scheduled on 70% or more.
Claims (6)
1. a kind of paramagnetic particle method extracts the kit of DNA, including digestion cracking system, articulated system, rinsing system, eluent system four
Big system.
2. a kind of paramagnetic particle method extracts the kit of DNA as described in claim 1, which is characterized in that the digestion cracking system packet
Include digestion buffer, protease, dithiothreitol (DTT).
3. a kind of paramagnetic particle method extracts the kit of DNA as described in claim 1, which is characterized in that the articulated system includes magnetic
Pearl and combine liquid, the magnetic bead be specified particle diameter and surface modification superparamagnetic nano particle, the combination liquid be magnetic bead inhale
Attached DNA provides low ph conditions with high salt, so that DNA is effectively combined with magnetic bead surfaces hydroxyl.
4. a kind of paramagnetic particle method extracts the kit of DNA as described in claim 1, which is characterized in that the rinsing system is ethyl alcohol
Solution.
5. a kind of paramagnetic particle method extracts the kit of DNA as described in claim 1, which is characterized in that the eluent system includes washing
De- liquid.
6. the kit extracting method that a kind of paramagnetic particle method extracts DNA, which is characterized in that
(1) it sample homogenization: to the type and proportion of chemical reagent in digestion buffer, and fullys shake and shakes up, guarantee system is equal
It is even, 10 minutes are stood under 25 degrees Celsius of room temperature after mixing；
(2) cell cracking: system being placed in 50 degrees Celsius of water-bath and cracked 20 to 30 minutes, and to system sustained oscillation,
Digestion buffer, protease, dithiothreitol (DTT) be digestion cracking process buffer condition is provided, digestion cracking process in DNA and
Separation of Proteins is released from nucleus；
(3) it is centrifuged: using independent centrifuge, maximum centrifugal force 1000g centrifugation time 2 to 5 minutes, is made by centrifugal action
It must be separated by solid-liquid separation, remove carrier and impurity；
(4) DNA is in conjunction with magnetic bead: magnetic bead is the superparamagnetic nano particle of specified particle diameter and surface modification, is magnetic bead in conjunction with liquid
Adsorption of DNA provides low ph conditions with high salt, so that DNA is effectively combined with magnetic bead surfaces hydroxyl, magnetic bead adsorbs in combining pendular ring border
DNA, the magnetic bead rate of recovery are stablized 95% or more；
(5) it washing: cleaning the soluble impurity of magnetic bead surfaces, main wash includes salt ion, protein and inhibition impurity,
The ingredient of rinsing liquid is ethanol solution, and washing times are 2 times；
(6) DNA is eluted: the eluent of 25 μ l is added, is uniformly mixed, the DNA that magnetic bead surfaces combine is eluted under low-salt conditions, point
The DNA that purifying can be collected from after is saved under conditions of subzero 20 to subzero 80 degrees Celsius, and DNA recovery efficiency is stablized
70% or more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811143843.1A CN109182331A (en) | 2018-09-29 | 2018-09-29 | A kind of paramagnetic particle method extracts the kit and extracting method of DNA |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811143843.1A CN109182331A (en) | 2018-09-29 | 2018-09-29 | A kind of paramagnetic particle method extracts the kit and extracting method of DNA |
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| CN109182331A true CN109182331A (en) | 2019-01-11 |
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| CN101613696A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | Extract the reagent of purify DNA |
| CN101935646A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting DNA from micro samples |
| CN102181434A (en) * | 2009-08-05 | 2011-09-14 | 公安部物证鉴定中心 | Method for extracting purified DNA from human exfoliative cells |
| CN102199594A (en) * | 2009-08-05 | 2011-09-28 | 公安部物证鉴定中心 | Method for extracting and purifying semen DNA |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
| CN103908945A (en) * | 2014-03-31 | 2014-07-09 | 洛阳惠尔纳米科技有限公司 | Preparation method of magnetic beads by extracting nucleic acid and application of method |
| US20160333339A1 (en) * | 2015-05-14 | 2016-11-17 | Longhorn Vaccines And Diagnostics, Llc | Rapid Methods for the Extraction of Nucleic Acids from Biological Samples |
| CN107779451A (en) * | 2017-11-20 | 2018-03-09 | 广州海思医疗科技有限公司 | A kind of mankind's dissociative DNA extracting method and its kit |
| CN108330125A (en) * | 2017-01-19 | 2018-07-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of bone or tooth DNA magnetic beads extracting method and reagent |
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2018
- 2018-09-29 CN CN201811143843.1A patent/CN109182331A/en not_active Withdrawn
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613696A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | Extract the reagent of purify DNA |
| CN102181434A (en) * | 2009-08-05 | 2011-09-14 | 公安部物证鉴定中心 | Method for extracting purified DNA from human exfoliative cells |
| CN102199594A (en) * | 2009-08-05 | 2011-09-28 | 公安部物证鉴定中心 | Method for extracting and purifying semen DNA |
| CN101935646A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting DNA from micro samples |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
| CN103908945A (en) * | 2014-03-31 | 2014-07-09 | 洛阳惠尔纳米科技有限公司 | Preparation method of magnetic beads by extracting nucleic acid and application of method |
| US20160333339A1 (en) * | 2015-05-14 | 2016-11-17 | Longhorn Vaccines And Diagnostics, Llc | Rapid Methods for the Extraction of Nucleic Acids from Biological Samples |
| CN108330125A (en) * | 2017-01-19 | 2018-07-27 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of bone or tooth DNA magnetic beads extracting method and reagent |
| CN107779451A (en) * | 2017-11-20 | 2018-03-09 | 广州海思医疗科技有限公司 | A kind of mankind's dissociative DNA extracting method and its kit |
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Application publication date: 20190111 |
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