CN104450683A - Method for extracting grape genome DNA from wine - Google Patents
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Abstract
The invention provides a method for extracting a grape genome DNA from wine. The method comprises the following steps: 1) DNA enrichment; 2) removal of polysaccharides; 3) crude extraction of DNA; 4) DNA purification; and 5) DNA recovery, thus obtaining the grape genome DNA. The DNA solution extracted by adopting the method is clear, the extraction and purification efficiency is high, and the obtained DNA has high concentration, good purity and good integrity.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of method extracting grape genomic dna from grape wine.
Background technology
Molecular Identification vinous comprises the extraction of grape DNA in grape wine and downstream molecular biology analyzes two parts, and from grape wine, wherein obtain high-quality grape DNA is the basis of carrying out downstream molecular biology analysis.Plant genome DNA extracting method is comparatively ripe, based on traditional methods such as CTAB method and SDS methods, is suitable for the DNA extraction method widespread use of dissimilar vegetable material, and even exploitation becomes commercial plant DNA extraction kit.But these DNA extraction method are not all suitable for the grape DNA extraction in grape wine.
Grape wine is height deep processed product, its production process goes through all too many levels such as fermentation, rolling bottle, ageing, clarification and filtration, in finished product grape wine, DNA content is atomic, and the chemical substance that a large amount of Polyphenols (tannin), polysaccharide, organic acid etc. introduced in grape wine by grape berry are complicated, have a strong impact on the molecular biological analysis in DNA extraction quality and downstream, have not yet to see a kind of grape wine DNA extraction method that can reach application level of report, also there is no ripe commercial kit.2000 so far, and the research report about DNA extraction and analysis in the grape wine of fruit juice, newly wine or ageing emerges in an endless stream.There are some researches show: even if by fermentation still there is the grape DNA of trace in grape wine, it is feasible for adopting molecular biology method to carry out by the grape DNA analysis in fruit juice or grape wine.From grape wine, extracting the enough and grape DNA of high-quality and filtering out the specific PCR primer of grape is the prerequisite that applied molecular biology means differentiate grape variety in grape wine, is also key point and the difficult point of the research of current grape wine gene authentication detection technology.After successfully extracting the grape DNA being suitable for carrying out downstream molecular biology experiment, the detection of grape specific gene can be carried out, to judge whether to brewage with original grape juice; Can microsatellite locus amplification be carried out, by the comparison of relevant grape microsatellite locus database, thus identify the grape variety in grape wine.
Summary of the invention
The object of the present invention is to provide a kind of method extracting grape genomic dna from grape wine, the method extraction purification efficiency is high, and the DNA concentration obtained is high, purity good, and the good integrity of tool.
The present invention is for reaching its object, and the technical scheme of employing is as follows:
From grape wine, extract a method for grape genomic dna, comprise the steps,
1) DNA enrichment: add 180 ~ 220 μ g glycogen, the beta-mercaptoethanol of 0.5 ~ 1.2v/v% and the Virahol of 0.7 ~ 1 times of volume in 20 ~ 30mL grape wine, after mixing, be placed in-65 DEG C ~-75 DEG C refrigerator 1 ~ 1.5h, then-18 DEG C ~-20 DEG C precipitation 12h ~ 24h are moved to, DNA in grape wine is precipitated, centrifugal afterwards, collecting precipitation; This step can be effectively remaining in enrichment grape wine grape DNA fragment, shorten the time needed for DNA enrichment.Due in grape wine containing more aldehydes matter, easy brownization, the more difficult removing of pigment, adopts this particular step, adds the reagent of consumption, can effectively avoid phenols to be oxidized, and helps DNA and sink agent.
2) removal of polysaccharide: to step 1) add 0.8 ~ 1mL in the precipitation that obtains and remove polysaccharide damping fluid, ice bath, mixing, centrifugal, abandon supernatant, again add polysaccharide damping fluid wherein, ice bath, mixing, centrifugal, abandon supernatant;
3) DNA slightly carries: 700 ~ 800 μ L adding 55 ~ 65 DEG C of preheatings wherein slightly carry damping fluid, mixing, and in 55 ~ 65 DEG C of water-bath 30 ~ 40min, period puts upside down mixing 3 ~ 4 times, then centrifugal, collects supernatant liquor;
4) DNA purifying: to step 3) in the supernatant liquor that obtains, add isopyknic phenol/chloroform/primary isoamyl alcohol mixed solution, centrifugal after mixing, collect supernatant liquor, in supernatant liquor, add RNase solution A carry out enzymolysis, then isopyknic chloroform/primary isoamyl alcohol mixed solution is added, centrifugal after mixing, get supernatant;
5) DNA reclaims, and obtains grape genomic dna.
Preferably, described step 1) in, after DNA enrichment precipitation, solution is 6,000g ~ 7,000g centrifugal 30min ~ 40min under 4 DEG C of conditions, collecting precipitation.
Preferably, described step 2) in, described goes polysaccharide damping fluid solvent to be water, and solute is the material of following final concentration: 80 ~ 120mmol/L pH value is Tris-HCl, 4 ~ 6mmol/L EDTA-Na of 8.0
2, 0.20 ~ 0.25mmol/L NaCl and mass volume ratio be the PVP-40T of 1.5 ~ 2.5%.Step 2) in add a certain amount of PVP can complexing polyphenol and terpene substances, polyphenol substance can be stoped to be oxidized to quinone, effectively can to prevent the pollution of polyphenol; Add the present invention and specifically remove polysaccharide damping fluid, can effectively remove pigment, polysaccharide and aldehydes matter, extracting solution obviously can become clarification.
Preferably, described step 2) in, the concrete steps that polysaccharide is removed are as follows:
A) first to be equipped with add 0.8mL ~ 1mL precooling in 50mL centrifuge tube that above-mentioned DNA precipitates remove polysaccharide damping fluid, precipitation on centrifugal tube wall is all rinsed, precipitation be all transferred in 2mL centrifuge tube, ice bath 5 ~ 8min, period puts upside down mixing 1 time;
B) by the mixed solution after ice bath in 4 DEG C, the centrifugal 5 ~ 8min of 12,000g ~ 13000g, abandon supernatant;
What in 50mL centrifuge tube, c) again add 0.5 ~ 0.8mL precooling removes polysaccharide damping fluid, precipitation is all transferred to step a) described in 2mL centrifuge tube, ice bath 5 ~ 8min after piping and druming mixing precipitation and solution, period puts upside down mixing 1 ~ 2 time;
D) mixed solution after ice bath, in 4 DEG C, the centrifugal 5 ~ 8min of 12,000g ~ 13000g, abandons supernatant.
Preferably, step 3) in, described damping fluid solvent of slightly carrying is water, the Tris-HCl that solute is the material of following final concentration: 18 ~ 22mmol/LEDTA, 8 ~ 12mmol/L pH value is 8.0,1.2 ~ 1.6mol/L NaCl, 1.8 ~ 2.2w/v%CTAB, 0.8 ~ 1.2v/v% mercaptoethanol, 1.8 ~ 2.2w/v%PVP-40T, 18 ~ 22mg/mL Proteinase K.In the weakly alkaline damping fluid of Tris-HCl-EDTA (pH 8.0), the CTAB of 1.8%-2.2% (w/v) coordinates 1.2 ~ 1.6mol/L NaCl effectively can extract DNA from grape wine.
Preferably, described step 3) in, centrifugal condition is: at 4 DEG C of centrifugal 8 ~ 10min of 12,000g ~ 13000g.
Preferably, step 4) described in DNA purifying carry out in accordance with the following steps:
A) isopyknic phenol/chloroform/primary isoamyl alcohol mixed solution is added, after vortex concussion mixing, the centrifugal 8 ~ 10min of 12,000g ~ 13000g under 4 DEG C of conditions, in described phenol/chloroform/primary isoamyl alcohol mixed solution, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1;
B) Aspirate supernatant is to another new 1.5mL centrifuge tube, add 5 ~ 8 μ L concentration be the RNase A of 10mg/mL in 37 DEG C of enzymolysis 20 ~ 30min, the concentration of described RNase A is 10mg/mL;
C), after adding isopyknic chloroform/primary isoamyl alcohol mixed solution vortex concussion mixing, under 4 DEG C of conditions, the centrifugal 8 ~ 10min of 12,000g ~ 13000g, gets supernatant, and in described chloroform/primary isoamyl alcohol mixed solution, the volume ratio of chloroform and primary isoamyl alcohol is 24:1.
After the cracking of DNA extraction damping fluid obtains and slightly carries DNA, with phenol/chloroform/primary isoamyl alcohol (25:24:1) and chloroform/primary isoamyl alcohol (24:1) gradation extracting, the impurity such as protein and phenols can be removed further.
Preferably, step 5) described in DNA reclaim carry out in accordance with the following steps:
A) to step 4) volumetric molar concentration that adds 0.1 times of volume in the supernatant liquor that obtains is the Virahol of the sodium acetate soln of 3 ~ 5mol/L, 5 ~ 8 μ g glycogen and 0.6 times of volume, in-18 DEG C ~-20 DEG C precipitation DNA 12 ~ 24h;
B) 12,000g ~ 13000 centrifugal 5 ~ 8min under 4 DEG C of conditions, collect DNA precipitation, abandon supernatant, use lavation buffer solution washing precipitation, afterwards 13,000g centrifugal 2min under 4 DEG C of conditions, collecting precipitation;
C) precipitate by the washing with alcohol of 75v/v%, then under 4 DEG C of conditions 12,000 ~ 13,000g centrifugal 2min, fling to ethanol, add 50 μ L Tris-EDTA water-bath 15min dissolving DNA at 65 DEG C.
Preferably, the pH value of described sodium acetate soln is 5.0 ~ 5.5.
Preferably, described lavation buffer solution contains the ethanol of 70 ~ 76v/v% and the ammonium acetate of 8 ~ 12mmol/L, and solvent is water.
Test-results shows, compared with prior art, the DNA solution clarification that present method extracts is bright, and extraction purification efficiency is high, and the DNA concentration obtained is high, purity good, and the good integrity of tool.
Accompanying drawing explanation
Fig. 1: red wine VvNCED gene real-time PCR detection result in embodiment 1;
Fig. 2: white wine VvNCED gene real-time PCR detection result in embodiment 2;
Fig. 3: the result figure that in embodiment 1, red wine VrZAG62 microsatellite locus is analyzed;
Fig. 4: the result figure that in embodiment 1, red wine MMD7 microsatellite locus is analyzed;
Fig. 5: the result figure that in embodiment 2, white wine VrZAG62 microsatellite locus is analyzed;
Fig. 6: the result figure that in embodiment 2, white wine MMD7 microsatellite locus is analyzed;
Fig. 7: 5 kinds of methods extract white wine VvNCED gene real-time PCR detection result, wherein, 1-positive control, 2-embodiment 2 is extracted, and 3 ~ 7 is method 1-4 extraction result (having overlap with negative control curve);
Fig. 8: 5 kinds of methods extract red wine VvNCED gene real-time PCR detection result, and wherein, 1-positive control, 2-embodiment 1 is extracted, and 3 ~ 7 is method 1-4 extraction result (having overlap with negative control curve).
Embodiment
Below in conjunction with drawings and Examples, technical scheme of the present invention is described further, but the present invention is not limited to following examples.
DNA in the commercially available red wine of embodiment 1 extraction purification
1, the DNA in the commercially available red wine of extraction purification
1), DNA enrichment
Get 30mL grape wine in the centrifuge tube of 50mL, add the Virahol of 220 μ g glycogen, 0.5% beta-mercaptoethanol (V/V) and 0.7 times of volume successively respectively, after abundant mixing, be placed in-75 DEG C of refrigerator 1h, then move to-18 DEG C and precipitate about 12h, to make in grape wine DNA precipitation, afterwards under 4 DEG C of conditions 7, the centrifugal 40min of 000g, collecting precipitation.
2) removal of polysaccharide
To step 1) add 0.8mL precooling in the precipitation that obtains remove polysaccharide damping fluid [80mmol/L Tris-HCl (pH8.0), 6m mol/L EDTA-Na
2, 0.25mmol/L NaCl, 1.5% (w/v) PVP-40T], with move liquid rob piping and druming the precipitation on centrifugal tube wall is all rinsed after, be all transferred in 2mL centrifuge tube, ice bath 5min, period puts upside down mixing 1 time as far as possible.
By 2mL centrifuge tube at 4 DEG C of 13,000g centrifugal 5min, abandon supernatant.
What in 50mL centrifuge tube, again add 0.5mL precooling removes polysaccharide damping fluid, robs piping and druming be again all transferred in 2mL centrifuge tube by the precipitation in centrifuge tube with moving liquid, and rob ice bath 5min after piping and druming mixing with moving liquid, period puts upside down mixing 1 ~ 2 time.
By 2mL centrifuge tube at 4 DEG C of 13,000g centrifugal 5min, abandon supernatant.
3) DNA slightly carries
The 700 μ L adding 65 DEG C of preheatings slightly carry damping fluid [18mmol/L EDTA, 8mmol/L Tris-HCl pH 8.0,1.2mol/LNaCl and 1.8% (w/v) CTAB, and add 0.8% (v/v) mercaptoethanol before use, 1.8% (w/v) PVP-40T, Proteinase K (18mg/mL)], vortex oscillation mixes, in 55 DEG C of water-bath 40min, period puts upside down mixing 3 times.At 4 DEG C of 12,000g centrifugal 10min, supernatant liquor is transferred to the 2mL centrifuge tube of another cleaning.
4) DNA purifying
Add equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1), after vortex concussion mixing, 13,000g centrifugal 8min under 4 DEG C of conditions.
Aspirate supernatant, to another new 1.5mL centrifuge tube, adds 5 μ L RNase A (10mg/mL) in 37 DEG C of enzymolysis 30min.
Add equal-volume chloroform: after primary isoamyl alcohol (24:1) vortex concussion mixing, 13,000g centrifugal 8min, get supernatant under 4 DEG C of conditions.
5) DNA reclaims
Add 0.1 times of volume sodium-acetate (3mol/L, pH=5), the Virahol of 5 μ g glycogen and 0.6 times of volume, be about 24h in-20 DEG C of overnight precipitation DNA.
13,000g centrifugal 5min under 4 DEG C of conditions, collect DNA precipitation, abandon supernatant, with lavation buffer solution (76% ethanol and 12mmol/L ammonium acetate) washing precipitation, and under 4 DEG C of conditions 13,000g centrifugal 2min.
Collecting precipitation, abandons supernatant, with 75% washing with alcohol precipitation, and under 4 DEG C of conditions 13,000g centrifugal 2min.
Air-dry or with nucleic acid vacuum-drying instrument dry ethanol, add 50 μ L TE, 65 DEG C of abundant dissolving DNAs of water-bath 15min.
DNA in the commercially available white wine of embodiment 2 extraction purification
1, the DNA in the commercially available white wine of extraction purification
1), DNA enrichment
Get 20mL grape wine in the centrifuge tube of 50mL, add the Virahol of 180 μ g glycogen, 1.2% beta-mercaptoethanol (V/V) and 1 times of volume successively respectively, after abundant mixing, be placed in-65 DEG C of refrigerator 1.5h, then move to-20 DEG C spend the night (about 24h), to make in grape wine DNA precipitation, afterwards under 4 DEG C of conditions 6, the centrifugal 30min of 000g, collecting precipitation.
2) removal of polysaccharide
To step 1) add 1mL precooling in the precipitation that obtains remove polysaccharide damping fluid [120mmol/L Tris-HCl (pH8.0), 4mmol/L EDTA-Na
2, 0.2mmol/L NaCl, 2.5% (w/v) PVP-40T)], with move liquid rob piping and druming the precipitation on centrifugal tube wall is all rinsed after, be all transferred in 2mL centrifuge tube, ice bath 8min, period puts upside down mixing 1 time as far as possible.
By 2mL centrifuge tube at 4 DEG C of centrifugal 8min of 12000g, abandon supernatant.
What in 50mL centrifuge tube, again add 0.8mL precooling removes polysaccharide damping fluid, robs piping and druming be again all transferred in 2mL centrifuge tube by the precipitation in centrifuge tube with moving liquid, and rob ice bath 8min after piping and druming mixing with moving liquid, period puts upside down mixing 1 ~ 2 time.
By 2mL centrifuge tube at 4 DEG C of centrifugal 8min of 12000g, abandon supernatant.
3) DNA slightly carries
The 800 μ L adding 55 DEG C of preheatings slightly carry damping fluid [22mmol/L EDTA, 12mmol/L Tris-HCl pH 8.0,1.6mol/L NaCl and 2.2% (w/v) CTAB, and add 1.2% (v/v) mercaptoethanol before use, 2.2% (w/v) PVP-40T, Proteinase K (22mg/mL)], vortex oscillation mixes, in 65 DEG C of water-bath 30min, period puts upside down mixing 3 ~ 4 times.At 4 DEG C of 12,000g centrifugal 10min, supernatant liquor is transferred to the 2mL centrifuge tube of another cleaning.
4) DNA purifying
Add equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1), after vortex concussion mixing, the centrifugal 10min of 12000g under 4 DEG C of conditions.
Aspirate supernatant, to another new 1.5mL centrifuge tube, adds 8 μ L RNase A (10mg/mL) in 37 DEG C of enzymolysis 20min.
Add equal-volume chloroform: after primary isoamyl alcohol (24:1) vortex concussion mixing, the centrifugal 10min of 12000g, gets supernatant under 4 DEG C of conditions.
5) DNA reclaims
Add 0.1 times of volume sodium-acetate (5mol/L, pH5.5), the Virahol of 8 μ g glycogen and 0.6 times of volume, be about 12h in-18 DEG C of precipitation DNA.
12000 centrifugal 8min under 4 DEG C of conditions, collect DNA precipitation, abandon supernatant, with lavation buffer solution (70% ethanol and 8mmol/L ammonium acetate) washing precipitation, and under 4 DEG C of conditions 13,000 centrifugal 2min.
Collecting precipitation, abandons supernatant, with 75% washing with alcohol precipitation, and under 4 DEG C of conditions 12,000g centrifugal 2min.
Air-dry or with nucleic acid vacuum-drying instrument dry ethanol, add 50 μ L TE (Tris-EDTA), 65 DEG C of abundant dissolving DNAs of water-bath 25min.
Embodiment 3 (the extraction result of embodiment 1,2 is detected)
3.1, DNA Concentration Testing
Carry out concentration determination with NANODROP1000 to the DNA extracted from grape wine, result is as shown in table 1 below:
Table 1 DNA extraction result
3.2, VvNCED2 gene amplification analysis
Select the primer of VvNCED gene and probe to carry out quantitative fluorescent PCR Establishing, primer and probe are synthesized by TaKaRa Bioisystech Co., Ltd.This is 94bp to primer extension product.
Its sequence is:
F：5’-ATGGCGACGGTATGGTTCA-3’，
R：5’-CGCTCCTGGACCAATCTCTG-3’，
Probe：Fam-AGTCACCCTTAATGGCGGTTC-Tamra。
VvNCED2 gene amplification analysis is carried out according to PCR reaction system as shown in table 1 below and condition.
Table 1 quantitative fluorescent PCR reaction system (20 μ L)
According to conventional real-time fluorescence PCR requirement, setting baseline and threshold value:
The DNA extracted using grape leave as positive reference, its equal Ct≤30 of VvNCED2 gene amplification result;
Using soybean as negative reference, design extraction blank and reagent blank in contrast, extract blank, reagent blank and negative reference VvNCED2 gene amplification result Ct ﹥ 45.
Red wine and equal Ct≤45 of white wine sample DNA VvNCED2 amplification, determine to utilize present method to extract and be applicable to the DNA of PCR.
Detected result as depicted in figs. 1 and 2.Wherein, Fig. 1 is grape VvNCED2 gene real-time PCR detection collection of illustrative plates in red wine.In figure, positive control is the fluorescence curve of grape leave, red wine is the fluorescence curve extracting DNA in red wine, negative control comprise the soybean as negative reference fluorescence curve, extract blank fluorescence curve and the fluorescence curve of DNA profiling blank.Fig. 2 is grape VvNCED2 gene real-time PCR detection collection of illustrative plates in white wine.In figure, positive control is the fluorescence curve of grape leave, white wine is the fluorescence curve extracting DNA in white wine, negative control comprise the soybean as negative reference fluorescence curve, extract blank fluorescence curve and the fluorescence curve of DNA profiling blank.As seen from the figure, extract the DNA obtained in red wine and occur amplification curve when carrying out grape VvNCED2 gene real-time fluorescence PCR, Ct value is 32, is less than 45, is positive findings, extract the DNA obtained in white wine and occur amplification curve when carrying out grape VvNCED2 gene real-time fluorescence PCR, Ct value is 33, is less than 45, is positive findings, this shows, all containing grape composition in red wine and white wine.Visible, utilize method of the present invention can extract effectively applicable DNA from red wine and white wine, and institute is carried in DNA containing grape composition.
3.3, microsatellite locus analysis
agents useful for same:
dNA 1000 reagent (5067-1504),
dNA chip,
pCR purification kit (28004),
universal PCR premixed liquid (2.0), 3mol/L sodium-acetate: pH 5.0.
sSR primer: as shown in table 2.
Table 2 SSR primer
sSR amplification and product reclaim purifying
1) PCR: adopt
pCR premixed liquid (2.0) carries out, and 50 μ L reaction systems, reaction system is as shown in table 3 below.
Table 3
Response procedures is: 95 DEG C, 5min; 94 DEG C, 5min; Annealing temperature (see table 3-4), 1min; 72 DEG C, 1min; 72 DEG C, 10min, cycle number is 45.
2) SSR amplified production reclaims
Employing Qiagen company
trace P CR purification kit (28004) carries out, and committed step is as follows:
According to the volume ratio of 5:1, PB damping fluid is joined in PCR reaction system mixed solution.
MinElute centrifugal column is put into the 2mL collection tube provided.
PCR primer is put into MinElute centrifugal column, centrifugal 1min, abandoned stream fluid, in the collection tube before being put back to by MinElute centrifuge tube.
The PE damping fluid of 750 μ L is added in MinElute centrifugal column, centrifugal 1min, abandoned stream fluid, in the collection tube before MinElute centrifuge tube is put back to.
Centrifugal column is put into the centrifugal 1min of 2mL collection tube.Effluent liquid must be abandoned before carrying out this and be centrifugal, the alcohol residue that PE damping fluid causes could be removed completely.
Each MinElute centrifugal column is loaded in clean 1.5ml centrifuge tube.
10 μ L EB damping fluid (10mM TrisCl, pH 8.5) or pure water are added, in order to eluted dna at MinElute centrifugal column Silicon moulds center.(note: Silicon moulds center must be aimed at when adding elution buffer, to guarantee the DNA of thorough elution of bound on film.) centrifugal column is left standstill 1min, centrifugal 1min afterwards.
The concentration of PCR primer after reclaiming with nucleic acid-protein analysis-e/or determining, is diluted to finite concentration by unified for concentration, is generally 50 ~ 100ng/ μ L.
3) microchip electrophoresis of SSR amplified production
Adopt agilent bio-analyser 2100 to carry out PCR and reclaim product analysis.
By the PCR primer after unified concentration according to the operation instruction of biological analyser 2100, design fabric swatch, analyze after application of sample, analytical results as illustrated in figures 3-6.
As seen from the figure, the grape DNA that present method extracts from grape wine may be used for microsatellite locus analysis.The peak type occurred is better, can tell the size of different strip segments accurately, illustrate that the extraction purification efficiency of present method is high, the DNA concentration obtained is high, the purity well also good integrity of tool.
Embodiment 4: compare with additive method
1. CTAB method (Pereira etc., 2011)
Carry out in accordance with the following steps: try to please and amass as 50mL plastic centrifuge tube, add the Virahol of 10mL wine samples and 0.7 times of volume, place 2 weeks in-20 DEG C, then 4,000g centrifugal 30min at ambient temperature, collecting precipitation;
Again gained precipitation is dissolved in Extraction buffer [the 20mmol/L EDTA of 850 μ L preheatings, 10mmol/L Tris-HCl pH 8.0,1.4mol/L NaCl and 2% (w/v) CTAB, and add 1% (v/v) mercaptoethanol before use, add 2% (w/v) PVP-40T subsequently, 20mg/mL Proteinase K], single vortex mixing, then bathes 60min by sample in 65 DEG C of temperature;
Then equal-volume 850 μ L phenol-chloroform-primary isoamyl alcohol mixed solution is added, 13,000g centrifugal 15min under 4 DEG C of conditions.Upper liquid is drawn 700 μ L and be transferred to a 2mL centrifuge tube, with 4 μ L in 37 DEG C of enzymolysis 30min;
The cold isopropanol adding 0.6 times of volume 420 μ L is about 24h in-20 DEG C of overnight precipitation DNA; After precipitation, in 4 DEG C of 10,000g centrifugal 15min, collect DNA precipitation;
Add 400 μ L TE (10mmol/L Tris-HCl, 1mmol/L EDTA, pH 8.0) dissolution precipitation, complete above-mentioned after, add 400 μ L phenol-chloroform-Virahol mixed solutions, mixing;
Finally 13,000g centrifugal 15min under 4 DEG C of conditions, then draw 300 μ L and are transferred to 1.5 centrifuge tubes, be about 24h with the cold isopropanol of 0.6 times of volume 180 μ L in-20 DEG C of overnight precipitation DNA by supernatant liquor; 10,000g centrifugal 15min under 4 DEG C of conditions, collect DNA precipitation, abandon supernatant, wash 5min with 100 μ L damping fluids (containing 76% ethanol and 10mM ammonium acetate).DNA is deposited in drying at room temperature, with 40 μ LTE wash-outs, be stored in-20 DEG C for subsequent use.
2. silica absorption method (Ye Yongzhong etc., 2006 (patent) CN 200610128466)
Carry out as follows:
A, to clean up with the Silica of pure water by 5g, add 5mL aqueous suspension Silica and precipitate, mixing, put 4 DEG C for subsequent use;
B, set up the grape wine of optimal adsorption condition
Magnetic stirring apparatus slowly adds lower series preparation and regulates grape wine: the NaOH solution 1) adding 10mol/L regulates pH vinous to be about 8.0; 2) CTAB to 1% (w/v) is added; 3) NaCl to 1.0mol/L is added; 4) mercaptoethanol to 0.2% (v/v) is added;
The absorption of C, grape wine DNA and recovery: (1) adds Silica suspension in above-mentioned grape wine mixed solution; Child care 1-2h at (2) 4 DEG C, constantly rocks, vibrates; (3) centrifugal, 5000g, 5min, 4 DEG C; (4) abandon supernatant liquor, precipitate 1-2 time with the washing fluid of short duration flushing Sliica containing 0.1mol/L Tris-HCl (pH8.0), 1%CTAB, 1mol/L NaCl; (5) centrifugal, 5000g, 5min, collect Silica precipitation; (6) add the pure water of equivalent to above-mentioned Silica precipitation, shake up, in 65 DEG C of hot eluted dnas of insulation 5-10min; (7) centrifugal (10000g, 5min) collects the supernatant liquor containing DNA; (8) supernatant liquor of equal-volume chloroform step (7), centrifugal 10000g, 3min, collect containing phase on DNA; (9) with DNA in the recovery of isopropanol precipitating method, concentrated supernatant; (10) the grape wine DNA reclaimed is dissolved in 50 μ L TE (10mmol/L Tris-HCl, pH 8.0) damping fluids.
3. SDS method (Nakamura etc., 2007)
Committed step comprises: be first precipitated centrifugal for grape wine, is suspended by precipitation Extraction buffer (100mmol/L Tris-HCl, 100mmol/L NaCl, 50mmol/L EDTA, 2%SDS, pH 8.5), 65 DEG C of water-bath 2h after vortex oscillation; Then the 2 mercapto ethanol adding 2% carries out incubation, phenol/chloroform/the primary isoamyl alcohol (25:24:1) adding 1 times of volume is centrifugal afterwards, be transferred to new Guan Zhonghou at supernatant liquor and add chloroform/primary isoamyl alcohol (24:1), then in supernatant liquor, add the isopropanol precipitating DNA of 0.6 times of volume; Centrifugal rear washing with alcohol 2 times, then precipitation is dissolved in 5mL TE, add incubation after RNase, add phenol/chloroform/primary isoamyl alcohol (25:24:1) again, after in upper water phase transition to new centrifuge tube, add the 3mol/L sodium acetate of 0.1 times of volume and the ethanol of 2 times of volumes, mixing postprecipitation spends the night.Again use washing with alcohol, after drying, be dissolved in 100 μ L TE.
4.
magnetic DNA Purification System for Food (food DNA purification system), reference reagent box operation instruction, committed step is as follows:
A. try to please and amass as 50mL plastic centrifuge tube, add the Virahol of 10mL wine samples and 0.7 times of volume, place 2 weeks in-20 DEG C, then 4,000g centrifugal 30min at ambient temperature, collecting precipitation.
B. add the Lysis Buffer A of 500 μ l and the RNase A of 5 μ L, lid upper tube cap, thermal agitation mixes; Add the Lysis Buffer B of 250 μ L, vortex shakes 10 – 15s and mixes, and builds pipe lid, keeps flat centrifuge tube, makes sample be placed in centrifuge tube side;
C. room temperature (22 – 25 DEG C) places 10min;
D. add 750 μ L Precipitation Solution, thermal agitation, fully suspends;
F.13, the centrifugal 10min of 000 × g;
G. supernatant liquor (liquid phase) is shifted to new 2mL centrifuge tube;
H. thermal agitation
pMPs bottle 15-30s, makes magnetic grain fully mix and suspend, and adds 50 μ L
pMPs bottle is to supernatant liquor, and violent vortex shakes.
I. add the Virahol of 0.8 times of volume, put upside down centrifuge tube 10-15min and mix, room temperature (22 – 25 DEG C) places 5min, and period is interrupted mixing, and sluicing pipe internal surface and magnetic grain.Pipe is placed in magnetic frame, leaves standstill 1min, suck supernatant liquor with liquid-transfering gun.
J. centrifuge tube is taken down from magnetic frame, add the Lysis Buffer B of 250 μ L, put upside down mixing 2-3min.Pipe is placed in magnetic frame again, after 1min, sucks supernatant.
K. centrifuge tube is taken down from magnetic frame, add 70% ethanol Eddy diffusion magnetic grain, pipe is placed in magnetic frame again, abandoning supernatant after standing 1min;
L. repeating step K twice (totally 3 times), sucks supernatant as far as possible with liquid-transfering gun;
M.. room temperature is placed 15 – 30min or 65 DEG C 10min and is made magnetic grain air-dry;
N. add 100 μ L TE or Nuclease-Free Water, vortex mixes, and in 65 DEG C of water-bath 5min.Pipe is placed in magnetic force
Frame 1min, is transferred to a new centrifuge tube meticulously to collect DNA by supernatant liquor.
Experimental result:
Adopt above-mentioned 4 kinds of existing DNA extraction method to carry out extraction that is white, red wine DNA respectively simultaneously.Table 4 and table 5 are depicted as grape extracting genome DNA product quality preliminary assessment in white wine and red wine.Found by the DNA solution visual observations extracted 5 kinds of methods, extract grape DNA in white wine by 5 kinds of methods and all can obtain clarifying bright DNA solution; But when extracting the grape DNA in red wine by 5 kinds of methods, the DNA solution adopting the embodiment 1 of the inventive method to extract is only had to be that clarification is bright, the DNA solution that all the other 4 kinds of methods extract is brown solution in various degree, illustrate aldehydes matter by brownization and pigment be not completely removed.
Visible by data in com-parison and analysis table, in grape wine, DNA concentration and purity are general lower, and differ greatly between differences method, from output and OD
260/ OD
280visible present method is optimum.Carried DNA is carried out VvNCED gene primer when carrying out quantitative fluorescent PCR qualitative detection, DNA in the white wine only having the inventive method to extract and red wine has amplified signal, the results are shown in Figure 7,8, illustrate and only have DNA quality and concentration in the obtained sample of the inventive method to be enough to carry out quantitative fluorescence analysis.
Table 4 Lung biopsy extracts DNA result in white wine
Table 5 Lung biopsy extracts DNA result in red wine
The above is only preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, any those skilled in the art, do not departing within the scope of technical solution of the present invention, the Equivalent embodiments changing or modify equivalent variations is made when the technology contents of above-mentioned announcement can be utilized, in every case be the content not departing from technical solution of the present invention, the any simple modification done above embodiment according to technical spirit of the present invention, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. from grape wine, extract a method for grape genomic dna, it is characterized in that, comprise the steps,
1) DNA enrichment: add 180 ~ 220 μ g glycogen, the beta-mercaptoethanol of 0.5 ~ 1.2v/v% and the Virahol of 0.7 ~ 1 times of volume in 20 ~ 30mL grape wine, after mixing, be placed in-65 DEG C ~-75 DEG C refrigerator 1 ~ 1.5h, then-18 DEG C ~-20 DEG C precipitation 12h ~ 24h are moved to, DNA in grape wine is precipitated, centrifugal afterwards, collecting precipitation;
2) removal of polysaccharide: to step 1) add 0.8 ~ 1mL in the precipitation that obtains and remove polysaccharide damping fluid, ice bath, mixing, centrifugal, abandon supernatant, again add polysaccharide damping fluid wherein, ice bath, mixing, centrifugal, abandon supernatant;
3) DNA slightly carries: 700 ~ 800 μ L adding 55 ~ 65 DEG C of preheatings wherein slightly carry damping fluid, mixing, and in 55 ~ 65 DEG C of water-bath 30 ~ 40min, period puts upside down mixing 3 ~ 4 times, then centrifugal, collects supernatant liquor;
4) DNA purifying: to step 3) in the supernatant liquor that obtains, add isopyknic phenol/chloroform/primary isoamyl alcohol mixed solution, centrifugal after mixing, collect supernatant liquor, in supernatant liquor, add RNase solution A carry out enzymolysis, then isopyknic chloroform/primary isoamyl alcohol mixed solution is added, centrifugal after mixing, get supernatant;
5) DNA reclaims, and obtains grape genomic dna.
2. method according to claim 1, is characterized in that, step 1) in, after DNA enrichment precipitation, solution is 6,000g ~ 7,000g centrifugal 30min ~ 40min under 4 DEG C of conditions, collecting precipitation.
3. method according to claim 1, is characterized in that, step 2) in, described goes polysaccharide damping fluid solvent to be water, and solute is the material of following final concentration: 80 ~ 120mmol/L pH value is Tris-HCl, 4 ~ 6mmol/L EDTA-Na of 8.0
2, 0.20 ~ 0.25mmol/L NaCl and mass volume ratio be the PVP-40T of 1.5 ~ 2.5%.
4. the method according to claim 1 or 3, is characterized in that, described step 2) in, the concrete steps that polysaccharide is removed are as follows:
A) first to be equipped with add 0.8mL ~ 1mL precooling in 50mL centrifuge tube that above-mentioned DNA precipitates remove polysaccharide damping fluid, precipitation on centrifugal tube wall is all rinsed, precipitation be all transferred in 2mL centrifuge tube, ice bath 5 ~ 8min, period puts upside down mixing 1 time;
B) by the mixed solution after ice bath in 4 DEG C, the centrifugal 5 ~ 8min of 12,000g ~ 13000g, abandon supernatant;
What in 50mL centrifuge tube, c) again add 0.5 ~ 0.8mL precooling removes polysaccharide damping fluid, precipitation is all transferred to step a) described in 2mL centrifuge tube, ice bath 5 ~ 8min after piping and druming mixing precipitation and solution, period puts upside down mixing 1 ~ 2 time;
D) mixed solution after ice bath, in 4 DEG C, the centrifugal 5 ~ 8min of 12,000g ~ 13000g, abandons supernatant.
5. method according to claim 1, it is characterized in that, step 3) described in damping fluid solvent of slightly carrying be water, solute is the material of following final concentration: 18 ~ 22mmol/L EDTA, 8 ~ 12mmol/L pH value be 8.0 Tris-HCl, 1.2 ~ 1.6mol/L NaCl, 1.8 ~ 2.2w/v%CTAB, 0.8 ~ 1.2v/v% mercaptoethanol, 1.8 ~ 2.2w/v%PVP-40T, 18 ~ 22mg/mL Proteinase K.
6. method according to claim 1 or 5, is characterized in that, described step 3) in, centrifugal condition is: at 4 DEG C of centrifugal 8 ~ 10min of 12,000g ~ 13000g.
7. method according to claim 1, is characterized in that, step 4) described in DNA purifying carry out in accordance with the following steps:
A) isopyknic phenol/chloroform/primary isoamyl alcohol mixed solution is added, after vortex concussion mixing, the centrifugal 8 ~ 10min of 12,000g ~ 13000g under 4 DEG C of conditions, in described phenol/chloroform/primary isoamyl alcohol mixed solution, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25:24:1;
B) Aspirate supernatant is to another new 1.5mL centrifuge tube, add 5 ~ 8 μ L concentration be the RNase A of 10mg/mL in 37 DEG C of enzymolysis 20 ~ 30min, the concentration of described RNase A is 10mg/mL;
C), after adding isopyknic chloroform/primary isoamyl alcohol mixed solution vortex concussion mixing, under 4 DEG C of conditions, the centrifugal 8 ~ 10min of 12,000g ~ 13000g, gets supernatant, and in described chloroform/primary isoamyl alcohol mixed solution, the volume ratio of chloroform and primary isoamyl alcohol is 24:1.
8. method according to claim 1, is characterized in that, step 5) described in DNA reclaim carry out in accordance with the following steps:
A) to step 4) volumetric molar concentration that adds 0.1 times of volume in the supernatant liquor that obtains is the Virahol of the sodium acetate soln of 3 ~ 5mol/L, 5 ~ 8 μ g glycogen and 0.6 times of volume, in-18 DEG C ~-20 DEG C precipitation DNA 12 ~ 24h;
B) 12,000g ~ 13000 centrifugal 5 ~ 8min under 4 DEG C of conditions, collect DNA precipitation, abandon supernatant, use lavation buffer solution washing precipitation, afterwards 13,000g centrifugal 2min under 4 DEG C of conditions, collecting precipitation;
C) precipitate by the washing with alcohol of 75v/v%, then under 4 DEG C of conditions 12,000 ~ 13,000g centrifugal 2min, fling to ethanol, add 50 μ L Tris-EDTA water-bath 15min dissolving DNA at 65 DEG C.
9. method according to claim 8, is characterized in that, the pH value of described sodium acetate soln is 5.0 ~ 5.5.
10. method according to claim 8 or claim 9, it is characterized in that, described lavation buffer solution contains the ethanol of 70 ~ 76v/v% and the ammonium acetate of 8 ~ 12mmol/L, and solvent is water.
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| CN104975008A (en) * | 2015-08-03 | 2015-10-14 | 江苏绿之邦生态庄园有限公司 | Method for extracting abundant total DNA (deoxyribonucleic acid) from grape fruits |
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| CN101210032A (en) * | 2006-12-26 | 2008-07-02 | 河南农业大学 | Simple and fast extraction method for DNA in grape wine |
| WO2011067630A1 (en) * | 2009-12-04 | 2011-06-09 | Universidade De Trás-Os-Montes E Alto Douro | Method and kit for dna extraction from vitis vinifera l. and for amplification and detection of grapevine varieties or cultivars in musts or wines |
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| CN101210032A (en) * | 2006-12-26 | 2008-07-02 | 河南农业大学 | Simple and fast extraction method for DNA in grape wine |
| WO2011067630A1 (en) * | 2009-12-04 | 2011-06-09 | Universidade De Trás-Os-Montes E Alto Douro | Method and kit for dna extraction from vitis vinifera l. and for amplification and detection of grapevine varieties or cultivars in musts or wines |
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| CN104975008A (en) * | 2015-08-03 | 2015-10-14 | 江苏绿之邦生态庄园有限公司 | Method for extracting abundant total DNA (deoxyribonucleic acid) from grape fruits |
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