CN101812444A - Blood genome magnetic bead small-amount extraction reagent kit and extraction method thereof - Google Patents
Blood genome magnetic bead small-amount extraction reagent kit and extraction method thereof Download PDFInfo
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- CN101812444A CN101812444A CN201010153890A CN201010153890A CN101812444A CN 101812444 A CN101812444 A CN 101812444A CN 201010153890 A CN201010153890 A CN 201010153890A CN 201010153890 A CN201010153890 A CN 201010153890A CN 101812444 A CN101812444 A CN 101812444A
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Abstract
The invention relates to a blood genome magnetic bead small extraction reagent kit and an extraction method thereof, which belong to the technical field of molecular biology. The reagent kit comprises seven ingredients: cell lysate, an RNA working solution, a cell protein precipitation liquid, self made magnetic beads, isopropanol, rinsing liquid and DNA eluate. The invention particularly aims at whole-blood materials with the small sample amount of 50 to 200 microliters, the whole-blood cells can be directly cracked without pretreatment on red cells, most hematoglobin and other impurities are removed through the cell protein precipitation liquid, then, the genome DNA is specifically absorbed by using monodispersing magnetic micro spheres, and the genome DNA on the magnetic micro spheres can be eluated after simple rinsing steps. The genome DNA extracted by the reagent kit has high yield, high purity and complete segment, and is applicable to the downstream tests such as PCR detection, cleavage reaction, nucleic acid hybridization and the like.
Description
Technical field
The invention belongs to technical field of molecular biology, a kind of blood genome magnetic bead small-amount extraction reagent kit and extracting method thereof particularly are provided, extract the test kit of poba gene group DNA fast in a small amount.
Technical background
DNA is most important biological information molecule as the carrier of genetic information, is the main object of molecular biology research.Extracting genomic dna is the basis of many molecular biology experiments, in order to carry out such as gene order-checking, in the routine operation tests such as the PCR of Southern hybridization (comprising RFLP), gene separates, structure BAC library, obtaining high molecular and highly purified genomic dna is very important prerequisite.Along with the deep development of Protocols in Molecular Biology, on gene level, disease is diagnosed basic fundamental means that become clinical diagnosis technology.Especially the individual recognition in the medical jurisprudence, paternity test etc., higher to the integrity and the purity requirement meeting of extracting genomic dna, because its genomic dna consumption is not high, develop quick a small amount of and extract the genomic dna test kit, just can reach the purpose of rapid detection.
Because it is convenient and reliable that blood is drawn materials, therefore complete genomic dna is normally extracted in above-mentioned research from blood.Briefly, extracting genome DNA has following principle: guarantee that 1, the DNA that extracts has certain length; 2, should not exist behind the purifying enzyme is had inhibiting material; 3, get rid of the pollution of organic solvent and metal ion; 4, protein, polysaccharide, lipid etc. are reduced to minimum level; 5, get rid of the pollution of other nucleic acid molecule.Present poba gene group extracting method has traditional phenol chloroform extraction method, proteolytic enzyme/SDS method and based on the Silica adsorption column method of medium and magnetic particle (magnetic bead) method or the like.Along with the use of commercialization DNA extraction test kit, especially can get fully and replace the extractive method of traditional phenol chloroform, and magnetic particle (magnetic bead) method is accepted extensively by increasing people with its exclusive advantage at the genome extraction of small sample quantities.
DNA extraction technology based on magnetic particle (magnetic bead) is the dna molecular that utilizes in hydrophilic magnetic particulate (magnetic bead) the adsorption sample solution, under adding the action of a magnetic field, the magnetic particle (magnetic bead) of adsorption of DNA is separated from sample solution, after suitably cleaning, add suitable solution and make DNA (magnetic bead) desorption from magnetic particle promptly obtain purified DNA.Extracting method based on magnetic particle (magnetic bead) makes the DNA extraction process greatly simplify, not only can in the test tube of routine, carry out with manual mode, also can in 96 holes or 382 hole microwell plates, carry out, obtain to use widely in American-European countries in the automatization mode.Magnetic particle (magnetic bead) method DNA extraction test kit will become the ideal auxiliary means of present molecular biology and clinic study with its simple and efficient to handle and time saving and energy saving characteristics.
Summary of the invention
The object of the present invention is to provide a kind of blood genome magnetic bead small-amount extraction reagent kit and extracting method thereof, extract the test kit of poba gene group DNA based on magnetic bead.Especially at the whole blood material of small sample quantities (50-200 microlitre), need not red corpuscle is carried out the direct cracking complete blood cell of pre-treatment, remove most of oxyphorase and other impurity by the cell protein precipitated liquid, utilize monodisperse magnetic microballoon specific adsorption genomic dna again, after only needing simple rinse step, just the genomic dna on the magnetic microsphere can be eluted.Good, the complete segment of genomic dna output height, purity that this test kit extracts is suitable for downstream tests such as PCR detection, endonuclease reaction, nucleic acid hybridization.
Test kit of the present invention comprises seven kinds of components such as cell pyrolysis liquid, RNA enzyme working fluid, cell protein precipitated liquid, magnetic bead, Virahol, rinsing liquid, DNA elutriant, and each component thes contents are as follows:
Cell pyrolysis liquid: 10-20mmol/L Tris (Tutofusin tris) salt, the SDS (sodium lauryl sulphate) of pH value 7.0-8.0,1-4mmol/L sequestrant, 50-100mmol/L Na salt, mass concentration 2-4% and the Proteinase K of final concentration 0.1-0.4mg/mL;
RNA enzyme working fluid: be ribonuclease A, its final concentration 10-20mg/mL;
Cell protein precipitated liquid: 5-8mol/L guanidinesalt, 0.5-2mol/L Na salt, 1-3mol/LKAC (Potassium ethanoate), 4-6mol/L HAC (acetic acid), pH value 5.0-6.0;
Magnetic bead: the monodisperse magnetic microballoon of particle diameter 1-2 μ m;
Virahol: analytically pure Virahol;
Rinsing liquid: 10-20mmol/L Tris (Tutofusin tris), pH value 8.0-9.0,100-200mmol/L Li salt, 1-4mmol/L sequestrant and dehydrated alcohol are by 1: the volume ratio of 3-5 mixes;
DNA elutriant: 10-20mmol/LTris (Tutofusin tris) salt, PH7.5-8.5.
The extracting method step of mentioned reagent box of the present invention is as follows:
Get the fresh blood 200 μ l that add antithrombotics, add lysis liquid 150-300 μ l, mixing, 55-70 ℃, 15min; Add 5-15 μ l RNA enzyme working fluid, place 5-10min for 25～35 ℃; Add cell albumen precipitation liquid: 250-400 μ l, turn upside down for several times, centrifugal 12000r/min, 3-5min presses close to the solution upper surface and carefully draws 350-700 μ l supernatant, is put in another centrifuge tube; Add 10-20 μ l magnetic bead, 200-400 μ l Virahol successively, horizontally rotate 1～1.5min; Magnetic separates 25～35s, and inhale and remove supernatant, rinse-added liquid 600-800 μ l, resuspended magnetic bead nucleic acid complexes cleans 2-3 time; Be placed on to inhale on the magnetic separator as far as possible and remove ethanol, room temperature～37 ℃ placement several minutes is until can't smell the alcohol smell; Add 50-80 μ l DNA elutriant, the 5-10min in ℃ water-bath to centrifuge tube in 55-65 is with complete wash-out genomic dna; Magnetic separates 30-40s, carefully draws supernatant in another centrifuge tube, standby or-20 ℃ of storages.
The present invention has the following advantages:
This test kit is easy and simple to handle, need not to red corpuscle carry out the direct cracking complete blood cell of pre-treatment, rapid extraction 1h gets final product complete operation;
This test kit does not use organic reagents such as phenol chloroform, and operator are had no side effect;
The genomic dna that this test kit extracts, under the guaranteed situation of purity, the DNA productive rate is higher, fragment is more complete.
Description of drawings
Fig. 1 is that two kinds of test kits extract the DNA agarose electrophoresis relatively.
Embodiment
Mouse, people's anticoagulated whole blood extracting genome DNA
Experiment material: gather fresh mouse, human blood sample 1mL, add an amount of antithrombotics EDTA sylvite, fully behind the mixing, standby in 4 ℃.
Extracting genome DNA step: get the fresh blood 200 μ l that add antithrombotics, add lysis liquid 200 μ l, mixing, 58 ℃, 15min; Add 10 μ l RNA enzyme working fluids, room temperature is placed 5-10min; Add cell albumen precipitation liquid: 350 μ l, turn upside down for several times, centrifugal 12000r/min, 3min presses close to the careful 350 μ l supernatants of drawing of solution upper surface, is put in another centrifuge tube; Add 20 μ l magnetic beads, 200 μ l Virahols successively, horizontally rotate 1min; Magnetic separates 30s, and inhale and remove supernatant, rinse-added liquid 750 μ l, resuspended magnetic bead nucleic acid complexes cleans 2 times; Be placed on to inhale on the magnetic separator as far as possible and remove ethanol, placed several minutes for 37 ℃, until can't smell the alcohol smell; Add 50 μ l DNA elutriants, to centrifuge tube 10min in 65 ℃ of water-baths, with complete wash-out genomic dna; Magnetic separates 30s, carefully draws supernatant in another centrifuge tube, standby or-20 ℃ of storages.
The test kit extraction step:
Get in the EP pipe of anticoagulated whole blood 100-250 μ l to 1.5ml, add the RBL Buffer of 4 times of volumes, the thorough mixing that turns upside down is even, incubated at room 5min.(2) hatch end after, the centrifugal 10min of 3000rpm; Abandon supernatant, leave and take bottom cell mass (red corpuscle of small amount of residual does not influence subsequent operations).(3) add 50 μ l Pre-Lysis buffer and fully blow and beat loosely with the rifle head, no visible cell is rolled into a ball (important) soon.(4) adding 600ul gDNA Extraction Buffer turns upside down and mixes, and room temperature is placed 10min, makes solution become transparence, and no visible cell is rolled into a ball fast, prolongs incubation time (important) in case of necessity.(5) above-mentioned solution is joined in the centrifugal post the centrifugal 5min of 13000rpm.(6) abandon liquid in the collection tube, in centrifugal post, add 500 μ l Washing Buffer (confirming to have added dehydrated alcohol), the centrifugal 1min of 13000rpm.(7) repeating step 6 once.(8) take out inner sleeve, discard liquid in the outer tube, still recover inner sleeve, do not add washing lotion, the 13000rpm sky is from 2min.(9) inner sleeve is moved in the new eppendorf pipe, room temperature leaves standstill 1min, makes the residual ethanol volatilization, adds Elution Buffer 40-100 μ l in film central authorities, and room temperature leaves standstill 1min, and 12, the centrifugal 2min of 000rpm obtains genomic dna.
Interpretation of result
Genomic dna output and the purity detecting extracted
Draw new mouse, the people's anticoagulated whole blood of gathering of 200 μ l respectively, use this test kit and A company test kit to carry out the extraction of genomic dna, the genomic dna that obtains is through the absorbance of UV spectrophotometer measuring 260nm and 280nm, and DNA purity is weighed (1.8-2.0), DNA concentration (μ g/mL)=OD260nm* extension rate * 50, DNA output=DNA concentration * elution volume with OD260nm/OD280nm ratio.
Two kinds of test kits of table 1. extract DNA result relatively
As can be seen from Table 1: two kinds of test kits extract on the genomic dna purity that obtains, all reached certain requirement, OD260nm/OD280nm ratio is all between 1.8-1.9, outline is better as a result for A company test kit, but the DNA output of gained of the present invention obviously improves, and is about 1.5 times of the test kit DNA of A company output.
The genomic DNA fragment integrity detection
Get the genomic dna that 2 μ l extract respectively, 100V voltage on 0.8% sepharose, electrophoresis 15min takes a picture with the ultraviolet gel imaging system, as shown in Figure 1.
As seen from Figure 1, two kinds of test kits extract the genomic DNA fragment that obtains all more than 23Kb, and A company test kit extracts about the about 30Kb of genomic DNA fragment that obtains, and this test kit extracts the genomic DNA fragment that obtains more than 50Kb.
Claims (2)
1. blood genome magnetic bead small-amount extraction reagent kit, test kit comprises cell pyrolysis liquid, RNA enzyme working fluid, cell protein precipitated liquid, magnetic bead, Virahol, rinsing liquid, seven kinds of components of DNA elutriant:
Cell pyrolysis liquid: 10-20mmol/L Tutofusin tris salt, the sodium lauryl sulphate of pH value 7.0-8.0,1-4mmol/L sequestrant, 50-100mmol/L Na salt, mass concentration 2-4% and the Proteinase K of final concentration 0.1-0.4mg/mL;
RNA enzyme working fluid: be ribonuclease A, its final concentration 10-20mg/mL;
Cell protein precipitated liquid: 5-8mol/L guanidinesalt, 0.5-2mol/LNa salt, 1-3mol/L Potassium ethanoate, 4-6mol/L acetic acid, pH value 5.0-6.0;
Magnetic bead: the monodisperse magnetic microballoon of particle diameter 1-2 μ m;
Virahol: analytically pure Virahol;
Rinsing liquid: the 10-20mmol/L Tutofusin tris, pH value 8.0-9.0,100-200mmol/L Li salt, 1-4mmol/L sequestrant and dehydrated alcohol are by 1: the volume ratio of 3-5 mixes;
DNA elutriant: 10-20mmol/L Tutofusin tris salt, PH8.0.
2. the extracting method of the described test kit of claim 1 is characterized in that, processing step is: get the fresh blood 200 μ l that add antithrombotics, add lysis liquid 150-300 μ l, mixing, 55-70 ℃, 15min; Add 5-15 μ l RNA enzyme working fluid, place 5-10min for 25～35 ℃; Add cell albumen precipitation liquid: 250-400 μ l, turn upside down for several times, centrifugal 12000r/min, 3-5min presses close to the solution upper surface and carefully draws 350-700 μ l supernatant, is put in another centrifuge tube; Add 10-20 μ l magnetic bead, 200-400 μ l Virahol successively, horizontally rotate 1～1.5min; Magnetic separates 25～35s, and inhale and remove supernatant, rinse-added liquid 600-800 μ l, resuspended magnetic bead nucleic acid complexes cleans 2-3 time; Be placed on to inhale on the magnetic separator as far as possible and remove ethanol, room temperature～37 ℃ placement several minutes is until can't smell the alcohol smell; Add 50-80 μ l DNA elutriant, the 5-10min in ℃ water-bath to centrifuge tube in 55-65 is with complete wash-out genomic dna; Magnetic separates 30-40s, carefully draws supernatant in another centrifuge tube, standby or-20 ℃ of storages.
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| 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 |
| CN102229925A (en) * | 2011-05-13 | 2011-11-02 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
| 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 |
| CN103205419A (en) * | 2013-04-25 | 2013-07-17 | 兰州美伯生物医药技术有限公司 | Method and kit for quickly separating and purifying DNA (Deoxyribonucleic Acid) of genome |
| CN103389241A (en) * | 2012-05-10 | 2013-11-13 | 中国科学院理化技术研究所 | Blood purifying method |
| CN103952397A (en) * | 2014-04-16 | 2014-07-30 | 天根生化科技（北京）有限公司 | Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead |
| CN105524917A (en) * | 2016-01-20 | 2016-04-27 | 苏州英芮诚生化科技有限公司 | Kit for extracting blood genome DNA based on magnetic bead method and use method for kit |
| CN105671030A (en) * | 2016-02-23 | 2016-06-15 | 苏州摩根基因科技有限公司 | Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method |
| CN105754994A (en) * | 2016-04-19 | 2016-07-13 | 武汉血液中心 | Method for extracting genomic DNA (deoxyribonucleic acid) from whole blood on basis of improved immunomagnetic bead method |
| CN108070585A (en) * | 2017-12-12 | 2018-05-25 | 杭州联川生物技术股份有限公司 | A kind of kit and its method based on magnetic bead technology extraction poba gene group DNA |
| CN108315325A (en) * | 2017-03-03 | 2018-07-24 | 绍兴迅敏康生物科技有限公司 | A kind of method and reagent for extracting nucleic acid substances using magnetic bead |
| CN109913445A (en) * | 2019-03-19 | 2019-06-21 | 宁波艾捷康宁生物科技有限公司 | One step washs paramagnetic particle method blood DNA extracts kit |
| CN111019940A (en) * | 2019-12-25 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof |
| CN112280838A (en) * | 2019-07-22 | 2021-01-29 | 苏州态和生物科技有限公司 | Method for removing PCR (polymerase chain reaction) pollution by using magnetic bead-nuclease filtration chromatographic column |
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2010
- 2010-04-23 CN CN201010153890A patent/CN101812444A/en active Pending
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| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| 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 |
| CN101935646B (en) * | 2010-09-13 | 2012-07-25 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting DNA from micro samples |
| CN101935645B (en) * | 2010-09-13 | 2012-07-25 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN102229925A (en) * | 2011-05-13 | 2011-11-02 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
| CN103389241A (en) * | 2012-05-10 | 2013-11-13 | 中国科学院理化技术研究所 | Blood purifying method |
| 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 |
| CN103205419A (en) * | 2013-04-25 | 2013-07-17 | 兰州美伯生物医药技术有限公司 | Method and kit for quickly separating and purifying DNA (Deoxyribonucleic Acid) of genome |
| CN103952397A (en) * | 2014-04-16 | 2014-07-30 | 天根生化科技（北京）有限公司 | Method for separating free nucleic acid from blood serum or blood plasma sample by using magnetic bead |
| CN105524917A (en) * | 2016-01-20 | 2016-04-27 | 苏州英芮诚生化科技有限公司 | Kit for extracting blood genome DNA based on magnetic bead method and use method for kit |
| CN105671030A (en) * | 2016-02-23 | 2016-06-15 | 苏州摩根基因科技有限公司 | Efficient plasma cell dissociation DNA extraction method based on paramagnetic particle method |
| CN105754994A (en) * | 2016-04-19 | 2016-07-13 | 武汉血液中心 | Method for extracting genomic DNA (deoxyribonucleic acid) from whole blood on basis of improved immunomagnetic bead method |
| CN108315325A (en) * | 2017-03-03 | 2018-07-24 | 绍兴迅敏康生物科技有限公司 | A kind of method and reagent for extracting nucleic acid substances using magnetic bead |
| CN108070585A (en) * | 2017-12-12 | 2018-05-25 | 杭州联川生物技术股份有限公司 | A kind of kit and its method based on magnetic bead technology extraction poba gene group DNA |
| CN109913445A (en) * | 2019-03-19 | 2019-06-21 | 宁波艾捷康宁生物科技有限公司 | One step washs paramagnetic particle method blood DNA extracts kit |
| CN112280838A (en) * | 2019-07-22 | 2021-01-29 | 苏州态和生物科技有限公司 | Method for removing PCR (polymerase chain reaction) pollution by using magnetic bead-nuclease filtration chromatographic column |
| CN111019940A (en) * | 2019-12-25 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof |
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Open date: 20100825 |