CN103013981A - Kit and method for extracting genome DNA of highly grain processed products by paramagnetic particle method - Google Patents
Kit and method for extracting genome DNA of highly grain processed products by paramagnetic particle method Download PDFInfo
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- CN103013981A CN103013981A CN2012105195312A CN201210519531A CN103013981A CN 103013981 A CN103013981 A CN 103013981A CN 2012105195312 A CN2012105195312 A CN 2012105195312A CN 201210519531 A CN201210519531 A CN 201210519531A CN 103013981 A CN103013981 A CN 103013981A
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Abstract
The invention discloses a kit and a method for extracting genome DNA of highly grain processed products by a paramagnetic particle method. The kit for extracting the genome DNA of highly grain processed products by the paramagnetic particle method comprises liquid A, liquid B, magnetic bead suspension, 75% of ethanol solution and TE solution, wherein the A liquid comprises 2-4wt% of CTAB (Cetyltrimethyl Ammonium Bromi), 1-1.5 mol/L of sodium chloride, 18-22 mol/L of EDTA (Ethylene Diamine Tetraacetic Acid), 80-120 mol/L of Tris-HCL, 10-30 mg/mL of protease K, 1-3wt% of beta- mercaptoethanol, and the pH of the A liquid is 7.5-8.5; the B liquid comprises 0.4-0.6wt% of CTAB and 0.03-0.05mol/L of sodium chloride, the pH of the B liquid is 7.5-8.5; the magnetic bead suspension comprises magnetic beads and 0.01-0.03wt% of NaN3 aqueous solution in a volume ratio of 1 to (1.5-2.5). The genome DNA extracted by the kit provided by the invention has a high recovery rate, high purity, complete segments, and no PCR (Polymerase Chain Reaction) inhibitors. The DNA extracting process including the overall process of cracking, purifying and collecting products can be completed by an apparatus, and the reagent which is used for the extracting is efficient and safe, so that the damages to the body can be avoided to the greatest extent.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of test kit and extracting method thereof that utilizes paramagnetic particle method to extract further processing of grains product gene group DNA.
Background technology
The Protocols in Molecular Biology of nucleic acid is to carry out the pathogenic microorganism examination, and transgenosis detects, and species are identified, one of the origin of species, diversity evaluation and the research means commonly used such as sibship, phyletic evolution thereof.The DNA of purifying carries out PCR to detect basic material, although in different samples (starting material, food ingredient, converted products), DNA is a metastable molecule, but for deep processed product, the DNA extractive technique remains the bottleneck based on the detection technique of DNA amplification, can extract high-quality dna molecular be to judge whether the analytical technology that adopts subsequently is reasonably crucial, and the sensitivity of extracting method, specificity also will be directly connected to the success or failure of follow-up test.For general analysis, good technology of preparing should be easy, safety, cheapness, and can guarantee DNA quality and quantity.Should possess certain concentration, purity, integrity for detection of the DNA that analyzes.Material and extractive technique all may be affected to these parameters, and conversely, also whether possibility impact analysis technology is reasonable for these parameters.
Traditional separate nucleic acid method mainly contain CTAB method, SDS method, PVP method and and silica gel adsorption, the step of extracting mainly comprises lysis, organic solvent extraction, precipitation, the processes such as high speed centrifugation, in the cleavage step, protein and polysaccharide are separated from the liquid phase of dissolving DNA in conjunction with CTAB and/or PVP, silica gel adsorption also can be used to assist extraction and the purifying of DNA, after the cracking, with activated nuclease and the PCR supressor in phenol and/or the chloroform removal liquid phase, such as lipophilic quasi-molecule, polysaccharide, protein.DNA precipitates in conjunction with the silica gel particle in Guanidinium hydrochloride-oxyhydroxide chaotropic agent, the precipitation washed with isopropyl alcohol.At last, precipitation is dissolved with low concentration salt solution.Related kit comprises Wizard test kit (Promega Corp., Madison, WI), Dneasy (Qiagen, Crawley, UK) etc.Numerous and diverse, the time-consuming length of the step of these purification process, the rate of recovery are low, and use the hazardous and noxious substances such as phenol, chloroform, Virahol in the leaching process, affect HUMAN HEALTH.
Magnetic strain separating and purifying technology is a kind of simple and effective nucleic acid purification method, the functional group that mainly connects energy specific adsorption nucleic acid at magnetic bead, in the cracked solution of sample, adsorb specifically target nucleic acid, by magnetic separation technique nucleic acid is extracted from cracked solution subsequently.Paramagnetic particle method can overcome the shortcoming of traditional nucleic acid method well, the characteristics such as tool is easy, quick, efficient, meet the nucleic acid automatization and extract requirement, therefore, the method has been widely used in the extraction of DNA of the tissue such as bacterium, animal blood, saliva, also correspondingly various test kits occurred.
But, compare with zooblast, vegetable cell is except having cell walls, also contain a large amount of polysaccharide, lipid, pigment and aldehydes matter, utilize existing paramagnetic particle method extraction DNA test kit that the genomic dna of plant tissue is extracted, the rate of recovery of products therefrom and purity are all lower, especially for the plant prod after the deep processing, because it is through after the pyroprocessing, the fracture palliating degradation degree of genomic dna is high, utilize existing paramagnetic particle method to extract the DNA test kit its genomic dna is extracted, the product rate of recovery and purity are all extremely low, can not satisfy the demand of further experiment.
Summary of the invention
For the existing deficiency of prior art, an object of the present invention is to provide the test kit that a kind of paramagnetic particle method extracts further processing of grains product gene group DNA.
Another object of the present invention provides the method that a kind of paramagnetic particle method extracts further processing of grains product gene group DNA.
The technical solution adopted in the present invention is:
A kind of paramagnetic particle method extracts the test kit of further processing of grains product gene group DNA, comprises A liquid, B liquid, magnetic bead suspension, 75% ethanolic soln and TE damping fluid, wherein,
A liquid contains 2～4wt%CTAB, 1～1.5mol/L sodium-chlor, 18～22mmol/L EDTA, 80～120 mmol/L Tris-HCL, 10～30 mg/mL Proteinase Ks, 1～3wt% beta-mercaptoethanol, pH=7.5～8.5;
B liquid contains 0.4～0.6 wt % CTAB, 0.03～0.05 mol/L sodium-chlor, pH=7.5～8.5;
The magnetic bead suspension is by the NaN of magnetic bead and 0.01～0.03wt%
3The volume ratio that the aqueous solution is pressed 1:1.5～2.5 forms.
Preferably, described A liquid contains 3 wt %CTAB, 1.4mol/L sodium-chlor, 20mmol/L EDTA, 100 mmol/L Tris-HCL, 20 mg/mL Proteinase Ks, 2 wt % beta-mercaptoethanols, pH=8.0.
Preferably, described B liquid contains 0.5 wt %CTAB, 0.04mol/L sodium-chlor, pH=8.0.
Preferably, the diameter of described magnetic bead is 3～7 μ m.
The extracting method of a kind of further processing of grains product gene group DNA comprises the steps
1) pretreated further processing of grains product is joined in the A liquid, 60～70 ℃ of temperature are bathed 40～80min;
2) get supernatant liquor, add B liquid and magnetic bead suspension, mix;
3) magnetic bead is transferred in 75% the ethanolic soln and cleaned;
4) magnetic bead after the cleaning carries out wash-out with the TE damping fluid, obtains the DNA of purifying.
The mass volume ratio of sample and A liquid is 1g:4～6ml.
The volume ratio of supernatant liquor and B liquid is 1:1～3.
The volume ratio of supernatant liquor and magnetic bead suspension is 10～20:1.
Described further processing of grains product comprises rice made products, feed, edible wet goods.
Beneficial effect of the present invention is:
1) the genomic dna rate of recovery of utilizing test kit of the present invention to extract is high, purity is high, complete segment, without the PCR inhibitory substance, use this reagent to extract 1g deep processed product ground rice, feed and 1000 mL peanut oil, purifying has obtained the approximately high purity genomic dna of 640 μ g, 520 μ g and 12 μ g respectively, between the purity A260/280=1.75-2.06.
2) DNA extraction method of the present invention only needs 4 steps, respectively cracking, precipitation, cleaning, wash-out, unattended operation, can be finished from the whole process of cracking, purifying, collection product by instrument, need not manual intervention and detection in the experimentation fully, and extract employed reagent highly effective and safe, at utmost avoid the injury to human body.
Description of drawings
Fig. 1 is the real-time fluorescence PCR detected result figure (1: test kit extraction ground rice DNA of the present invention that different methods extracts ground rice, feed genomic dna, 2: test kit of the present invention extracts feedstuff DNA, 3: commercially available reagent box 1 extracts ground rice DNA, 4: commercially available reagent box 1 extracts feedstuff DNA, 5: commercially available reagent box 2 extracts ground rice DNA, 6: commercially available reagent box 2 extracts feedstuff DNA, 7: commercially available reagent box 3 extracts ground rice DNA, 8: commercially available reagent box 3 extracts feedstuff DNA);
Fig. 2 is the genomic dna fluorescent PCR amplification figure of Oleum Arachidis hypogaeae semen extraction.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, but be not limited to this.
Embodiment 1
Paramagnetic particle method extracts the test kit of further processing of grains product gene group DNA, comprises A liquid, B liquid, magnetic bead suspension, 75% ethanolic soln and TE damping fluid, wherein,
Consisting of of A liquid: 3wt%CTAB, 1.4mol/L sodium-chlor, 20mmol/L EDTA, 100 mmol/L Tris-HCL, 20 mg/mL Proteinase Ks, the 2wt% beta-mercaptoethanol, pH=8.0, solvent is water;
Consisting of of B liquid: 0.5wt%CTAB, 0.04mol/L sodium-chlor, pH=8.0, solvent is water;
Consisting of of TE damping fluid: 10mM Tris-HCl, 1mM EDTA, pH=8.0, solvent are water;
Consisting of of magnetic bead buffer solution: magnetic bead (diameter is 3～7 μ m) (OMEGA company) and 0.02wt% NaN
3The aqueous solution is pressed the volume ratio of 1:2 and is mixed.
Utilize the mentioned reagent box to extract ground rice or feed genomic dna, comprise the steps:
1. get ground rice or feed that 1g pulverized, join in the 5ml A liquid, 65 ℃ of hatching 60min.
2. normal temperature 10000rpm, centrifugal 10min is until precipitation fully, is drawn supernatant liquor for subsequent use.
3. shift 300 μ l supernatant liquors, join first and second hole of KingFisher mL 5 unions, add 600 μ l B liquid and 20 μ l magnetic bead suspensions in the first hole; Add 600 μ l B liquid in the second hole; The third and fourth hole adds 75% ethanolic soln of 900 μ l; The 5th hole adds 400 μ L TE damping fluids.
4. 5 unions are put into Full automatic instrument for extracting nucleic acid Thermo Scientific KingFisher mL, changed over to magnetosheath, working procedure, programstep is as follows:
(1) first hole vortex mixing 60 seconds;
(2) the absorption magnetic bead is transferred to the second hole, vortex mixing 60 seconds;
(3) the absorption magnetic bead is transferred in 75% ethanol of the 3rd hole vortex mixing 120 seconds;
(4) the absorption magnetic bead is transferred in 75% ethanol of the 4th hole vortex mixing 120 seconds;
(5) be transferred to and adsorb magnetic bead, drying at room temperature 10min on the magnetic frame;
(6) discharge in magnetic bead to the five hole TE damping fluids the resuspended magnetic bead of vortex.
5. draw the 5th hole TE damping fluid, the genomic dna that namely extracts places refrigerator for subsequent use;
6. use micro-spectrophotometer (Nanodrop-1000) to measure concentration and the purity of DNA, with the extraction result of other 3 kinds of conventional commercially available reagent box in contrast, the result is as shown in table 1.
7. the genomic dna that extracts being carried out real-time fluorescence PCR detects, ground rice detects paddy rice native gene GOS9, and feed detects soybean lectin plain gene LECTIN, and primer and probe sequence see Table 2, with the extraction result of other 3 kinds of conventional commercially available reagent box in contrast, experimental result is seen Fig. 1.
The test kit of employing embodiment 1 extracts the genomic dna in the peanut oil, comprises the steps:
1. get 200 ml samples oil, in 500 mL beakers, add isopyknic sterilized water, 20-25 ℃ in stirring more than 1 hour on the magnetic stirring apparatus; Then in 12000r/min, centrifugal 15-20min carefully draws lower floor's water; Add 200 ml samples oil at aqueous phase, repeat 1-2 step 5 time.
2. carefully water is transferred in the culture dish, culture dish is put in-80 ℃ of freeze overnight, then culture dish is gone in the Vacuumdrier to moisture and evaporate fully; Add 1 mL A liquid to culture dish, 65 ℃ of temperature were bathed 4 hours.
3. shift respectively 300 μ l supernatant liquors, join first and second hole of KingFisher mL 5 unions, add 600 μ l B liquid and 20 μ l magnetic bead suspensions in the first hole; Add 600 μ l B liquid in the second hole; The third and fourth hole adds the ethanolic soln of 900 μ l 75%; The 5th hole adds 50 μ L TE damping fluids.
4. 5 unions are put into Full automatic instrument for extracting nucleic acid Thermo Scientific KingFisher mL, changed over to magnetosheath, working procedure, programstep is as follows:
(1) first hole vortex mixing 60 seconds;
(2) the absorption magnetic bead is transferred to the second hole, vortex mixing 60 seconds;
(3) the absorption magnetic bead is transferred in 75% ethanol of the 3rd hole vortex mixing 120 seconds;
(4) the absorption magnetic bead is transferred in 75% ethanol of the 4th hole vortex mixing 120 seconds;
(5) be transferred to and adsorb magnetic bead, drying at room temperature 10min on the magnetic frame;
(6) discharge in magnetic bead to the five hole TE damping fluids the resuspended magnetic bead of vortex.
5. draw the 5th hole TE damping fluid, the genomic dna that namely extracts places refrigerator for subsequent use.
6. use micro-spectrophotometer (Nanodrop-1000) to measure concentration and the purity of DNA, the result is as shown in table 2.
7. the genomic dna that extracts is carried out real-time fluorescence PCR and detect peanut chitinase gene chi2.2, the primer is HS-F and HS-R, and probe is that HS-P(sees Table 2).Extract peanut genome as positive control with the inventive method, deionized water is as negative control, every group establish two parallel.The result as shown in Figure 2.
Experimental result shows, DNA extraction test kit of the present invention is compared with the commercially available reagent box of other 3 kinds of routines, high to the genomic dna rate of recovery, purity is high, complete segment, without the PCR inhibitory substance.In addition, test kit of the present invention also can be used for the extraction of genomic dna in the edible oil.
Only for introducing preferred case of the present invention, to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention all should be regarded as a part of the present invention to above embodiment.
＜110〉Zhuhai Entry-Exit Inspection and Quarantine Bureau of P.R.C.
＜120〉a kind of paramagnetic particle method extracts test kit and the method for further processing of grains product gene group DNA
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Claims (9)
1. the test kit of a paramagnetic particle method extraction further processing of grains product gene group DNA comprises A liquid, B liquid, magnetic bead suspension, 75% ethanolic soln and TE damping fluid, wherein,
A liquid contains 2～4wt%CTAB, 1～1.5mol/L sodium-chlor, 18～22mmol/L EDTA, 80～120 mmol/L Tris-HCL, 10～30 mg/mL Proteinase Ks, 1～3wt% beta-mercaptoethanol, pH=7.5～8.5;
B liquid contains 0.4～0.6 wt % CTAB, 0.03～0.05 mol/L sodium-chlor, pH=7.5～8.5;
The magnetic bead suspension is by the NaN of magnetic bead and 0.01～0.03wt%
3The volume ratio that the aqueous solution is pressed 1:1.5～2.5 forms.
2. test kit according to claim 1 is characterized in that, described A liquid contains 3wt%CTAB, 1.4mol/L sodium-chlor, 20mmol/L EDTA, 100 mmol/L Tris-HCL, 20mg/mL Proteinase K, 2 wt % beta-mercaptoethanols, pH=8.0.
3. test kit according to claim 1 is characterized in that, described B liquid contains 0.5%CTAB, 0.04mol/L sodium-chlor, pH=8.0.
4. test kit according to claim 1 is characterized in that, the diameter of described magnetic bead is 3～7 μ m.
5. the extracting method of a further processing of grains product gene group DNA comprises the steps:
1) pretreated further processing of grains product is joined in claim 1 or the 2 described A liquid, 60～70 ℃ of temperature are bathed 40～80min;
2) get supernatant liquor, add claim 1 or 3 described B liquid and magnetic bead suspension claimed in claim 1, mix;
3) magnetic bead is transferred in 75% the ethanolic soln and cleaned;
4) magnetic bead after the cleaning carries out wash-out with the TE damping fluid, obtains the DNA of purifying.
6. extracting method according to claim 5 is characterized in that, in the step 1), the mass volume ratio of sample and A liquid is 1g:4～6ml.
7. extracting method according to claim 5 is characterized in that step 2) in, the volume ratio of supernatant liquor and B liquid is 1:1～3.
8. extracting method according to claim 5 is characterized in that step 2) in, the volume ratio of supernatant liquor and magnetic bead suspension is 10～20:1.
9. extracting method according to claim 5 is characterized in that, described further processing of grains product comprises rice made products, feed, edible wet goods.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480369A (en) * | 2021-12-24 | 2022-05-13 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
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| 曾富华主编: "《生物化学实验技术教程》", 31 July 2011, article "8.1植物基因组DNA和质粒DNA的提取及回收", pages: 184-187 * |
| 赵卫东 等: "植物源性食品DNA提取方法的建立及几种方法比较", 《食品安全与检测》, vol. 37, no. 9, 30 September 2012 (2012-09-30), pages 306 - 310 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480369A (en) * | 2021-12-24 | 2022-05-13 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
| CN114480369B (en) * | 2021-12-24 | 2022-12-27 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
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