CN1660879A - Method for separating residual DNA from soy sauce, soybean milk and tomato ketchup - Google Patents
Method for separating residual DNA from soy sauce, soybean milk and tomato ketchup Download PDFInfo
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Abstract
A process for separating residual DNA from soy, soybean milk and tomato sass includes centrifugal treating, taking deposit, dissolving in CTAB solution, waving, adding phenol or chloroform, stirring, centrifugal separation, applying supernatant to sephadex-G50 column, adding isopropanol, centrifugal separation, washing deposit with alcohol, centrifugal separation, washing, drying, adding Chelax solution, stirring, boiling, and centrifugal separation. Its supernatant can be directly used as the template of PCR.
Description
Technical field
The present invention is a biological technical field, relate in particular to a kind of from soy sauce, soymilk, tomato-sauce the method for DNA isolation.
Background technology
Since nineteen eighty-three, the 1st strain transgenic plant were born in the world, be the modern biotechnology fast development of core with the transgenic technology.With genetically modified organism is that the food of direct food or raw material processing is genetically modified food.Present most of genetically modified food derives from transgenic crop, and its contribution to the mankind is very big, can improve quality, disease and insect resistance, the volume increase of food and reduce the agricultural chemicals usage quantity, thereby bring remarkable economic efficiency and social benefit.At back to nature, advocate today of Organic food, the human consumer still suspects to the security of genetically modified food, and the arguement that genetically modified crops and food are brought is that people are quite unexpected.Bone of contention: the one, genetically modified food is to the direct or indirect influence of HUMAN HEALTH; The 2nd, genetically modified crops are to the Nature ecological balance damage; The 3rd, the ethics morals problem of being correlated with.Because the potential risk of genetically modified organism and products thereof, its management is subjected to the great attention of national governments.The label of genetically modified food is the problem of dispute maximum at present.The World Health Organization has proposed genetically modified food is carried out the requirement of safety evaluation in the nineties in 20th century.On January 28th, 2000, international community is on the basis that meets each other half way, problems such as transboundary movement with regard to some major issues, especially transgenic technology and products thereof of Biosafety have been reached common recognition, have passed through " Katki Na Biosafety Protocol " at Montreal, CAN.The international safety standard of genetically modified food is being prepared to work out by the international food council that stipulates under the Food and Argriculture OrganizationFAO and the World Health Organization, world other countries and area have also been worked out relevant law or rules one after another.Because various countries limit to some extent to each other genetically modified food and food raw material trade, more external in addition food companies abandoned the genetically modified food raw material need not, then adopt the non-transgenic raw material.Be protection consumers in general's rights and interests, satisfy its preference, right to know and for the needs of international trade, methods for detecting transgenic foods more and more causes the attention of national governments and related food supervisory organ.But because the complicacy and the diversity of gene recombination technology have been brought very big difficulty to methods for detecting transgenic foods, therefore set up the accurate detection method of international standard, become national governments and investigator's problem demanding prompt solution.
From transgenic technology foundation, transgenic technology is also set up thereupon, because in the technology of gene recombination, be unable to do without the detection and the screening of recombination.The specific aim of these methods is very strong, and what have is only effective to the structure of certain genetically modified organism, but lays the foundation for the foundation of transgenosis detection technique.Mainly contain two class transgenic detection methods at present: based on the pulsating PCR of specific DNA (polymerase ChainReaction) detection technique with based on ELISA (the Enzyme-linked Immunosorbent Assays) detection method of specific proteins.The former comprises exogenous promoter, terminator, reporter gene and goal gene at the specific DNA segment.Latter's specific proteins mainly is exogenous object protein and the expressed albumen of reporter genes.The starting point difference of two kinds of methods respectively has relative merits.In general, the PCR method can be with DNA amplification to 100 remaining in the food more than ten thousand times, detect highly sensitively, but misoperation easily produces the false positive phenomenon.The ELISA method is not suitable for the food through high temperature process, can't detect because heating can make protein denaturation.The Meyer Rolf of German Bornstein university in 1996 etc. has proved the possibility that PCR detects genetically modified food.Up to the present, existing more than the 30 kind of transgenic product in the whole world obtains the safety evaluation of concerned countries, the overwhelming majority contains CaMV 35S promoter and NOS terminator in existing commercialization transgenic product, two kinds of factors are usually used in the structure of transgenic plant, and this just provides convenience for people set up at the transgenosis sun screening detection method of these two kinds of compositions.Some external laboratories are with reporter gene GUS, NPE II gene recently, and goal gene CRY, CPT etc. strive detecting more accurately and reliably transgene component as detecting target.Because round pcr requires relatively stricter, high sensitivity and breadboard carryover contamination false positive results often occurs in daily genetically modified food detects.In genetically modified food detects mainly be at present with 35S CaMV promotor and NOS terminator as detecting target, judge whether contain transgene component in the food.Qualitative screening PCR itself has limitation, the PCR method of being taked because of its hypersensitivity often with the false positive phenomenon, and operational error adds that some reaction inhibition factors also can bring false negative phenomenon, and the deficiency of its maximum is to carry out quantitative analysis to transgene component.For this reason, investigators have been developed different quantitative PCR detecting methods on the basis of qualitative screening PCR method.At present, comparatively sophisticated abroad method mainly contain quantitative competitive PCR (Quantitativecompetitive, QC-PCR) and Real-time PCR method.
The research of gene chip just is being devoted in present some laboratories in the world, and this is a very promising developing direction of transgenosis detection technique.The principle of biochip technology is according to sophisticated situ hybridization technology in the molecular biology, i.e. the base pairing of DNA and sequence principle of complementarity, and very large amount probe molecule is fixed in about 1cm
2On the chip of size, hybridize with the sample of mark then, judge the quantity of target molecule in the sample by the power that detects hybridization signal.This technology can be carried out check and analysis to a large amount of DNA or RNA molecule, improves detection efficiency and accuracy greatly.In a word, the development trend of genetically modified food detection technique should be easy and simple to handle, expense is lower, universality is strong.Existing or emerging genetically modified food can both quick and conveniently be detected exactly.
Generally speaking, the security of genetically modified organism must be based upon on the result of transgene component detection.Transgenic detection method commonly used in the world at present mainly contains two kinds: the one, and it is that source structure genetic expression goes out the protein detection method that protein is purpose in addition that detection has or not from the other biological composition; Another kind is the DNA detection method of the detection promotor that non-transgenic plant did not have, terminator, marker gene.Because protein detection method transgenic product low to content and deep processing can't detect, so DNA detection method range of application is wide relatively.Now, be raw material to adopting transgenic product both at home and abroad, even adopt the DNA detection method, also can't detect transgene component through deep processing or accurately machined food.For example: making of soy sauce, salad oil will be by fermentation, heating with operation such as separate, the foreign DNA of importing and the protein of generation thereof decompose.Do not judge whether to contain transgene component so also there is effective detection method up to now, this is a generally acknowledged difficult problem at international organic sphere.
Because transgenic product becomes the starting material of food more and more,, therefore import starting material and food are carried out the outstanding problem that transgenosis detects becomes genetically modified organism security detection as soybean, corn etc.If can from test sample, extract the high-quality tracer level DNA that is suitable for pcr template, from those deep processings or accurately machined food (soy sauce, salad oil, soymilk, tomato-sauce, biscuit etc.), extract the high-quality DNA of tracer level especially, be still the thorny problem that transgenosis safe detects.Given this, the present invention has set up a kind of from soy sauce, soymilk, the method of separating residual DNA in the tomato-sauce, on the basis of conventional DNA extraction technology, having increased Sephadex-G 50 posts separates and two steps of Chelex solution thermal treatment, can not only effectively remove soy sauce, soymilk, small molecules tunning in the tomato-sauce, polysaccharide, sanitas, fixing agent, material such as alum and pigment, and can obtain the high-quality tracer level DNA that can be used for detecting, this method is simple and effective, cost is low, sensitivity and recall rate height, the tracer level DNA that is obtained can be used for qualitative and quantitative pcr amplification.
Summary of the invention
An object of the present invention is to provide a kind of from soy sauce, soymilk, tomato-sauce the method for separating residual DNA.
The step of method is:
1) get soy sauce, soymilk, the tomato-sauce of 50ml, 12, the centrifugal 15-20min of 000rpm abandons supernatant, keeps precipitation;
2) get 0.5ml bromohexadecane base TMA (TriMethylAmine) and extract the damping fluid dissolution precipitation, it is 2% bromohexadecane base TMA (TriMethylAmine) that bromohexadecane base TMA (TriMethylAmine) is extracted damping fluid, 50mmol/L Tris alkali, pH8.0, the 10mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0,0.8mol/L sodium-chlor;
3) 65 ℃ of insulation 30～90min, the light and slow frequently therebetween mixing of putting upside down; Add 1: 1 phenol/chloroform of 0.5ml, mixing is to the milkiness shape, and 12, the centrifugal 10-15min of 000rpm is to phase-splitting, draws supernatant liquor with the centrifugal post of crossing of Sephadex-G50 post;
4) dehydrated alcohol that adds isopyknic Virahol or 2.5 times of volumes was placed 1-2 hour in-70 ℃ or-20 ℃, and 12, the centrifugal 10-15min of 000rpm abandons supernatant, keeps precipitation;
5) 70% washing with alcohol precipitation, 12, the centrifugal 10-15min of 000rpm, repeated washing precipitation 1-2 time after the drying at room temperature, adds the Chelex solution of 200 μ l 10% in precipitation, abundant mixing, 100 ℃ are boiled 5-10min;
6) 12, after 000rpm was centrifugal, its supernatant can be directly used in the template of polymerase chain reaction.
Advantage of the present invention is: 1) in conventional DNA extraction technology, increasing Sephadex-G 50 posts separates and two steps of Chelex solution thermal treatment, so can effectively remove the materials such as small molecules tunning, polysaccharide, sanitas, fixing agent, alum and pigment in soy sauce, soymilk, the tomato-sauce, and obtain the high-quality tracer level DNA that can be used for detecting; 2) can effectively remove the PCR reaction DNA that supressor obtained its sensitivity and recall rate in polymerase chain reaction (PCR) amplification and be higher than conventional DNA extraction method.
3) this method is simple effectively, cost is low.
Description of drawings
Fig. 1 .lectin gene PCR amplification, among the figure: M is a molecular weight marker, 1 is the soybean positive control, 2 is the pcr amplification product of test kit extracting DNA, 3 is the pcr amplification product of CTAB method extracting DNA, 4 is the pcr amplification product of the direct boiling method extracting of Chelex DNA, and 5 is the pcr amplification product of method extracting DNA of the present invention, 6 negative contrasts;
Fig. 2 .35S-CTP gene PCR amplification, among the figure: M is a molecular weight marker, 1 is the soybean positive control, 2 is the pcr amplification product of test kit extracting DNA, 3 is the pcr amplification product of CTAB method extracting DNA, 4 is the pcr amplification product of the direct boiling method extracting of Chelex DNA, and 5 is the pcr amplification product of method extracting DNA of the present invention, 6 negative contrasts;
Fig. 3 .Cp4-epsps gene PCR amplification, among the figure: M is a molecular weight marker, 1 is the soybean positive control, 2 is the pcr amplification product of test kit extracting DNA, 3 is the pcr amplification product of CTAB method extracting DNA, 4 is the pcr amplification product of the direct boiling method extracting of Chelex DNA, and 5 is the pcr amplification product of method extracting DNA of the present invention, 6 negative contrasts;
Fig. 4 .Nos terminator gene PCR amplification, among the figure: M is a molecular weight marker, 1 is the soybean positive control, 2 is the pcr amplification product of test kit extracting DNA, 3 is the pcr amplification product of CTAB method extracting DNA, 4 is the pcr amplification product of the direct boiling method extracting of Chelex DNA, and 5 is the pcr amplification product of method extracting DNA of the present invention, 6 negative contrasts;
Fig. 5 .CaMV35S promoter gene pcr amplification result, among the figure: M is a molecular weight marker, 1 is the soybean positive control, 2 is the pcr amplification product of test kit extracting DNA, 3 is the pcr amplification product of CTAB method extracting DNA, 4 is the pcr amplification product of the direct boiling method extracting of Chelex DNA, and 5 is the pcr amplification product of method extracting DNA of the present invention, 6 negative contrasts.
Embodiment
Reagent used in the present embodiment is the domestic reagent except that chloroform, ethanol, primary isoamyl alcohol, Virahol, and all the other reagent are the promega product.
(1) CTAB method extracting residual DNA
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.The bromohexadecane base TMA (TriMethylAmine) (CTAB) that pulverous dry-matter is added 400 μ l precoolings extract damping fluid I (50mmol/L Tris-Cl, 10mmol/L EDTA, 800mmol/LNaCl, pH8.0) in.The CTAB that adds 65 ℃ of preheatings of 500 μ l extracts damping fluid II (2% CTAB, 50mmol/LTris-Cl, 10mmol/L EDTA, 800mmol/L NaCl, pH8.0), 1 μ l 2 mercapto ethanol, mixing, 65 ℃ of insulation 30-90min, the light and slow frequently therebetween mixing of putting upside down.Add 450 μ l chloroform/primary isoamyl alcohol after waiting to be chilled to room temperature, light and slow mixing to the solution of putting upside down is the milkiness shape.The centrifugal 2min of 12000rpm is to phase-splitting.Supernatant liquor is transferred in the clean centrifuge tube, adds 5 μ l Rnase A liquid storages, be incubated 30min under the room temperature.Add 600 μ l Virahols and 60 μ l sodium acetate solutions successively, the light and slow mixing of putting upside down.12, the centrifugal 10min of 000rpm, deposit D NA.After abandoning supernatant liquor, add 800 μ l, 76% ethanol, 12, the centrifugal 10min of 000rpm, abandon supernatant liquor after, add 100 μ l, 70% washing with alcohol precipitation again, 12, the centrifugal 5min of 000rpm, deposit D NA removes residual ethanol.The DNA resolution of precipitate is in 100 μ l TE damping fluids, and 4 ℃ of preservations are standby.
(2) the direct boiling method of Chelex
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.Get the Chelex solution dissolution precipitation of 200 μ l 10%; Vibration number min fully is dissolved in the Chelex solution precipitation, and 100 ℃ are boiled 5～10min, and 12, the centrifugal 5min of 000rpm, supernatant liquor promptly can be used for PCR.
(3) test kit method extracting residual DNA
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.Add the 25ml normal hexane, behind the thorough mixing after 2 hours, add 2.5ml Buffer A after, heat in 65 ℃ of water-baths again, and constantly mix, after 2 hours with 12, the centrifugal 10min of 000rpm makes organic phase and water separately, and the water intaking phase also adds 40 μ l (500ng/ μ l) salmon sperm dna.Add and the isopyknic Buffer B of supernatant, mixing, after normal temperature is placed 10min, 12, the centrifugal 5min of 000g abandons supernatant, keeps precipitation; In precipitation, add 60 μ l Buffer C, after using the rifle head with its thorough mixing (very important) behind 37 ℃ of placement 2min, in 37 ℃ of dissolution precipitations, add 300 μ l Buffer D behind the 5min, turn upside down behind the abundant mixing 10 times, take out centrifugal post, centrifugal post is placed on the sleeve pipe of a 2ml, solution is joined in the centrifugal post, place 2min; Centrifugal post and 2ml sleeve pipe one are reinstated 8000g after centrifugal 30 seconds, abandon the solution in the 2ml sleeve pipe, add 200 μ l Wash Buffer I in centrifugal post, 8000g discards solution after centrifugal 30 seconds; Add 200 μ l Wash Buffer II (please adding ethanol according to the prompting on the reagent bottle before using for the first time) in centrifugal post, 8000g discards solution after centrifugal 30 seconds; Add 200 μ l Wash Buffer II in centrifugal post, 8000g discards solution after centrifugal 30 seconds; 12, centrifugal 30 seconds of 000g is to remove traces of residual solution in the centrifugal post; Centrifugal post is placed in the new 1.5ml centrifuge tube, nets the centrifugal column bottom careful 50 μ l Elution Buffer of adding of central authorities, behind 37 ℃ of placement 2min, 12, centrifugal 30 seconds of 000g; If need to improve yield, the available centrifugal 50 μ l Elution Buffer that get off carry out wash-out again, promptly 37 ℃ place 2min after, 12, centrifugal again 30 seconds of 000g; Solution in the centrifuge tube promptly can be used as the template of PCR reaction, advises PCR reaction use 2 μ l DNA, and all the other sample retention are at-20 ℃.
(4) " molecular cloning " method extracting residual DNA
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.Adding 20ml extraction damping fluid in the 50ml centrifuge tube (100mmol/L TrisCl, pH8.0,20mmol/L EDTA, pH8.0,500mmol/L NaCl, 1.5%SDS), 60 ℃ of water-bath preheatings.Pour into immediately in the centrifuge tube of preheating above-mentioned dry-matter added the liquid nitrogen pulverize in mortar after, acutely shake mixing, 60 ℃ of water bath heat preservation 30-60min (time is long, DNA output height) shake frequently.Add 20ml chloroform/amylalcohol/ethanolic soln, put upside down mixing (need the band gloves, prevent injured skin), leave standstill 5-10min under the room temperature, make water and organic phase layering (in case of necessity mixing) again.The centrifugal 5min of 5000rpm under the room temperature.Carefully pipette supernatant liquor to another 50ml centrifuge tube, add 1 times of volume Virahol, mixing is placed 30min under the room temperature, with the centrifugal 5min of 5000rpm, will precipitate in the immigration TE pipe again.The precipitation of Shou Jiing like this, often difficulty is dissolved in TE, can place more than the 15min 60 ℃ of water-baths, to help dissolving.With the centrifugal 5min of dna solution 3000rpm, supernatant liquor is poured clean 5ml centrifuge tube into.Add 5 μ l RnaseA (10 μ g/ μ l), 37 ℃ of 10min remove RNA (RNA can omit this step to the general nothing influence of operation, analysis of DNA).The ice ethanol that adds the 3mol/L NaAc and the 2 * volume of 1/10 volume, mixing, place about 20min for-20 ℃, 12, the centrifugal 10min of 000rpm, 70% ethanol rinsing heavily is dissolved in 1mlTE damping fluid (10mmo/L TrisCl (pH8.0) with DNA, 1mmol/L EDTA (pH8.0)) ,-20 ℃ of storages.
(5) method of the present invention proposes residual DNA from soy sauce, soymilk, tomato-sauce
1) get soy sauce, soymilk, the tomato-sauce of 50ml, 12, the centrifugal 15min of 000rpm abandons supernatant, keeps precipitation;
2) get 0.5ml bromohexadecane base TMA (TriMethylAmine) and extract the damping fluid dissolution precipitation, it is 2% bromohexadecane base TMA (TriMethylAmine) that bromohexadecane base TMA (TriMethylAmine) is extracted damping fluid, 50mmol/L Tris alkali, pH8.0, the 10mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0,0.8mol/L sodium-chlor;
3) 65 ℃ of insulation 30min, the light and slow frequently therebetween mixing of putting upside down; Add 0.5ml phenol/chloroform (1: 1), the light and slow mixing of putting upside down is to the milkiness shape, and 12, the centrifugal 10min of 000rpm is to phase-splitting, draws supernatant liquor with the centrifugal post of crossing of Sephadex-G 50 posts;
4) add isopyknic Virahol (or dehydrated alcohol of 2.5 times of volumes) and placed 1 hour in-70 ℃ (or-20 ℃), 12, the centrifugal 10min of 000rpm abandons supernatant, keeps precipitation;
5) 70% washing with alcohol precipitation, 12, the centrifugal 10min of 000rpm, repeated washing precipitation 1-2 time after the drying at room temperature, adds the Chelex solution of 200 μ l 10% in precipitation, abundant mixing, 100 ℃ are boiled 5min;
6) 12, after 000rpm was centrifugal, its supernatant can be directly used in the template of polymerase chain reaction.
(6) polymerase chain reaction (PCR) amplification
The total reaction system is 50 μ l, the template DNA of the different sources that the above-mentioned four kinds of methods of 10 μ l are extracted, 5 μ l, 10 * PCR reaction buffer (500mmol/L KCl, 100mmol/L TrisCl, under 25 ℃, pH9.0,1.0% Triton X-100), 4 μ l 25mmol/L MgCl
2, 4 kinds of dNTP of 4 μ l (every kind of 2.5mmol/L), 0.5 μ l upstream primer (100ng/ μ l), 0.5 μ l downstream primer (100ng/ μ l), 1 μ l Taq archaeal dna polymerase (5U/ μ l), redistilled water 25 μ l, behind the mixing centrifugal 5 seconds.With mixture 94 ℃ down ice-cold behind the heating 5min, the rapid centrifugal several seconds, make that drop is sink to the pipe end on the tube wall, add Taq archaeal dna polymerase (the about 2.5U of 0.5 μ l), centrifugal slightly behind the mixing.95 ℃ of sex change 5min, with 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 amplified reaction circulations, carry out PCR.After last took turns loop ends, insulation 10min under 72 ℃ made the reaction product amplification fully.
The primer of pcr amplification is respectively:
1.lectin gene
Lec-F1：5’GCCCTCTACTCCACCCCCATCC3’
Lec-R1：5’GCCCATCTGCAAGCCTTTTTGTG3’
2.35S-CTP gene
SC-F1：5’TGATGTGATATCTCCACTGACG3’
SC-R1：5’TGTATCCCTTGAGCCATGTTGT3’
3.Cp4-epsps gene
CE-F1：5’CCTTCATGTTCGGCGGTCTCG3’
CE-R1：5’GCGTCATGATCGGCTCGATG3’
4.Nos terminator gene
Pnos-F1：5’GAATCCTGTTGCCGGTCTTG3’
Pnos-R1：5’TTATCCTAGTTTGCGCGCTA3’
5.CaMV35S promoter gene
35S-F：5’GCTCCTACAAATGCCATCATTGC3’
35S-R：5’GATAGTGGGATTGTGCGTCATCCC3’
(7) agarose gel electrophoresis
(1mmol/L EDTA pH8.0), prepares 1% sepharose liquid for 45mmol/L Tris alkali, 45mmol/L boric acid with 0.5 * TBE electrophoretic buffer; Add 10mg/ml ethidium bromide (EB) solution mother liquor, making its final concentration is 0.5 μ g/ml; Prepare corresponding gel slab with the glue plate; Get 10 μ l PCR products and 2 μ l, 6 * load sample damping fluid (0.25% tetrabromophenol sulfonphthalein, 40% (w/v) aqueous sucrose solution) mixing, carefully add with the micropipette rifle in the sample cell of gel slab; Control voltage remains on 60-80V, and electric current is more than 40mA, and the demand working power supply carries out electrophoresis; When the tetrabromophenol sulfonphthalein band moves to apart from the about 2cm in gel forward position, stop electrophoresis; Be to observe dyeing under the long wavelength ultraviolet lamp of 254nm and take pictures at wavelength.
(1) CTAB method extracting residual DNA
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.The CTAB that pulverous dry-matter is added 400 μ l precoolings extract damping fluid I (50mmol/L Tris-Cl, 10mmol/L EDTA, 800mmol/L NaCl, pH8.0) in.The CTAB that adds 65 ℃ of preheatings of 500 μ l extracts damping fluid II (2% CTAB, 50mmol/L Tris-Cl, 10mmol/L EDTA, 800mmol/L NaCl, pH8.0), 1 μ l 2 mercapto ethanol, mixing, 65 ℃ of insulation 30-90min, the light and slow frequently therebetween mixing of putting upside down.Add 450 μ l chloroform/primary isoamyl alcohol after waiting to be chilled to room temperature, light and slow mixing to the solution of putting upside down is the milkiness shape.The centrifugal 2min of 12000rpm is to phase-splitting.Supernatant liquor is shifted as in the clean centrifuge tube, add 5 μ l Rnase A liquid storages, be incubated 30min under the room temperature.Add 600 μ l Virahols and 60 μ l sodium acetate solutions successively, the light and slow mixing of putting upside down.12, the centrifugal 10min of 000rpm, deposit D NA.After abandoning supernatant liquor, add 800 μ l, 76% ethanol, 12, the centrifugal 10min of 000rpm, abandon supernatant liquor after, add 100 μ l, 70% washing with alcohol precipitation again, 12, the centrifugal 5min of 000rpm, deposit D NA removes residual ethanol.The DNA resolution of precipitate is in 100 μ l TE damping fluids, and 4 ℃ of preservations are standby.
(2) the direct boiling method of Chelex
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.Get the Chelex solution dissolution precipitation of 200 μ l 10%; Vibration number min fully is dissolved in the Chelex solution precipitation, and 100 ℃ are boiled 5～10min, and 12, the centrifugal 5min of 000rpm, supernatant liquor promptly can be used for PCR.
(3) test kit method extracting residual DNA
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.Add the 25ml normal hexane, behind the thorough mixing after 2 hours, add 2.5ml Buffer A after, heat in 65 ℃ of water-baths again, and constantly mix, after 2 hours with 12, the centrifugal 10min of 000rpm makes organic phase and water separately, and the water intaking phase also adds 40 μ l (500ng/ μ l) salmon sperm dna.Add and the isopyknic Buffer B of supernatant, mixing, after normal temperature is placed 10min, 12, the centrifugal 5min of 000g abandons supernatant, keeps precipitation; In precipitation, add 60 μ l Buffer C, after using the rifle head with its thorough mixing (very important) behind 37 ℃ of placement 2min, in 37 ℃ of dissolution precipitations, add 300 μ l Buffer D behind the 5min, turn upside down behind the abundant mixing 10 times, take out centrifugal post, centrifugal post is placed on the sleeve pipe of a 2ml, solution is joined in the centrifugal post, place 2min; Centrifugal post and 2ml sleeve pipe one are reinstated 8000g after centrifugal 30 seconds, abandon the solution in the 2ml sleeve pipe, add 200 μ l Wash Buffer I in centrifugal post, 8000g discards solution after centrifugal 30 seconds; Add 200 μ l Wash Buffer II (please adding ethanol according to the prompting on the reagent bottle before using for the first time) in centrifugal post, 8000g discards solution after centrifugal 30 seconds; Add 200 μ l Wash Buffer II in centrifugal post, 8000g discards solution after centrifugal 30 seconds; 12, centrifugal 30 seconds of 000g is to remove traces of residual solution in the centrifugal post; Centrifugal post is placed in the new 1.5ml centrifuge tube, nets the centrifugal column bottom careful 50 μ l Elution Buffer of adding of central authorities, behind 37 ℃ of placement 2min, 12, centrifugal 30 seconds of 000g; If need to improve yield, the available centrifugal 50 μ l Elution Buffer that get off carry out wash-out again, promptly 37 ℃ place 2min after, 12, centrifugal again 30 seconds of 000g; Solution in the centrifuge tube promptly can be used as the template of PCR reaction, advises PCR reaction use 2 μ l DNA, and all the other sample retention are at-20 ℃.
(4) " molecular cloning " method extracting residual DNA
Get 50ml soy sauce, soymilk, tomato-sauce, through 12, the centrifugal 10min of 000rpm abandons supernatant liquor, keeps the extracting that precipitation is used for DNA; Or get dry-matter behind 80 ℃ of heating evaporation moisture and be used for DNA extraction; Or get dry-matter after the lyophilize and be used for DNA extraction.Adding 20ml extraction damping fluid in the 50ml centrifuge tube (100mmol/L TrisCl, pH8.0,20mmol/L EDTA, pH8.0,500mmol/L NaCl, 1.5%SDS), 60 ℃ of water-bath preheatings.Pour into immediately in the centrifuge tube of preheating above-mentioned dry-matter added the liquid nitrogen pulverize in mortar after, acutely shake mixing, 60 ℃ of water bath heat preservation 30-60min (time is long, DNA output height) shake frequently.Add 20ml chloroform/amylalcohol/ethanolic soln, put upside down mixing (need the band gloves, prevent injured skin), leave standstill 5-10min under the room temperature, make water and organic phase layering (in case of necessity mixing) again.The centrifugal 5min of 5000rpm under the room temperature.Carefully pipette supernatant liquor to another 50ml centrifuge tube, add 1 times of volume Virahol, mixing is placed 30min under the room temperature, with the centrifugal 5min of 5000rpm, will precipitate in the immigration TE pipe again.The precipitation of Shou Jiing like this, often difficulty is dissolved in TE, can place more than the 15min 60 ℃ of water-baths, to help dissolving.With the centrifugal 5min of dna solution 3000rpm, supernatant liquor is poured clean 5ml centrifuge tube into.Add 5 μ l RnaseA (10 μ g/ μ l), 37 ℃ of 10min remove RNA (RNA can omit this step to the general nothing influence of operation, analysis of DNA).The ice ethanol that adds the 3mol/L NaAc and the 2 * volume of 1/10 volume, mixing, place about 20min for-20 ℃, 12, the centrifugal 10min of 000rpm, 70% ethanol rinsing heavily is dissolved in 1mlTE damping fluid (10mmo/L TrisCl (pH8.0) with DNA, 1mmol/L EDTA (pH8.0)) ,-20 ℃ of storages.
(5) method of the present invention is extracted residual DNA
1) get soy sauce, soymilk, the tomato-sauce of 50ml, 12, the centrifugal 20min of 000rpm abandons supernatant, keeps precipitation;
2) get 0.5ml bromohexadecane base TMA (TriMethylAmine) and extract the damping fluid dissolution precipitation, it is 2% bromohexadecane base TMA (TriMethylAmine) that bromohexadecane base TMA (TriMethylAmine) is extracted damping fluid, 50mmol/L Tris alkali, pH8.0, the 10mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0,0.8mol/L sodium-chlor;
3) 65 ℃ of insulation 90min, the light and slow frequently therebetween mixing of putting upside down; Add 0.5ml phenol/chloroform (1: 1), the light and slow mixing of putting upside down is to the milkiness shape, and 12, the centrifugal 15min of 000rpm is to phase-splitting, draws supernatant liquor with the centrifugal post of crossing of Sephadex-G 50 posts;
4) add isopyknic Virahol (or dehydrated alcohol of 2.5 times of volumes) and placed 2 hours in-70 ℃ (or-20 ℃), 12, the centrifugal 15min of 000rpm abandons supernatant, keeps precipitation;
5) 70% washing with alcohol precipitation, 12, the centrifugal 15min of 000rpm, repeated washing precipitation 1-2 time after the drying at room temperature, adds the Chelex solution of 200 μ l 10% in precipitation, abundant mixing, 100 ℃ are boiled 10min;
6) 12, after 000rpm was centrifugal, its supernatant can be directly used in the template of polymerase chain reaction.
(6) polymerase chain reaction (PCR) amplification
The total reaction system is 50 μ l, the template DNA of the different sources that the above-mentioned four kinds of methods of 10 μ l are extracted, 5 μ l, 10 * PCR reaction buffer (500mmol/L KCl, 100mmol/L TrisCl, under 25 ℃, pH9.0,1.0% Triton X-100), 4 μ l 25mmol/L MgCl
2, 4 kinds of dNTP of 4 μ l (every kind of 2.5mmol/L), 0.5 μ l upstream primer (100ng/ μ l), 0.5 μ l downstream primer (100ng/ μ l), 1 μ l Taq archaeal dna polymerase (5U/ μ l), redistilled water 25 μ l, behind the mixing centrifugal 5 seconds.With mixture 94 ℃ down ice-cold behind the heating 5min, the rapid centrifugal several seconds, make that drop is sink to the pipe end on the tube wall, add Taq archaeal dna polymerase (the about 2.5U of 0.5 μ l), centrifugal slightly behind the mixing.95 ℃ of sex change 5min, with 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, carry out 35 amplified reaction circulations, carry out PCR.After last took turns loop ends, insulation 10min under 72 ℃ made the reaction product amplification fully.
The primer of pcr amplification is respectively:
1.lectin gene
Lec-F1：5’GCCCTCTACTCCACCCCCATCC3’
Lec-R1：5’GCCCATCTGCAAGCCTTTTTGTG3’
2.35S-CTP gene
SC-F1：5’TGATGTGATATCTCCACTGACG3’
SC-R1：5’TGTATCCCTTGAGCCATGTTGT3’
3.Cp4-epsps gene
CE-F1：5’CCTTCATGTTCGGCGGTCTCG3’
CE-R1：5’GCGTCATGATCGGCTCGATG3’
4.Nos terminator gene
Pnos-F1：5’GAATCCTGTTGCCGGTCTTG3’
Pnos-R1：5’TTATCCTAGTTTGCGCGCTA3’
5.CaMV35S promoter gene
35S-F：5’GCTCCTACAAATGCCATCATTGC3’
35S-R：5’GATAGTGGGATTGTGCGTCATCCC3’
(7) agarose gel electrophoresis
(1mmol/L EDTA pH8.0), prepares 1% sepharose liquid for 45mmol/L Tris alkali, 45mmol/L boric acid with 0.5 * TBE electrophoretic buffer; Add 10mg/ml ethidium bromide (EB) solution mother liquor, making its final concentration is 0.5 μ g/ml; Prepare corresponding gel slab with the glue plate; Get 10 μ l PCR products and 2 μ l, 6 * load sample damping fluid (0.25% tetrabromophenol sulfonphthalein, 40% (w/v) aqueous sucrose solution) mixing, carefully add with the micropipette rifle in the sample cell of gel slab; Control voltage remains on 60-80V, and electric current is more than 40mA, and the demand working power supply carries out electrophoresis; When the tetrabromophenol sulfonphthalein band moves to apart from the about 2cm in gel forward position, stop electrophoresis; Be to observe dyeing under the long wavelength ultraviolet lamp of 254nm and take pictures at wavelength.
Claims (2)
1. the method for a separating residual DNA from soy sauce, soymilk, tomato-sauce.It is characterized in that the step of method is:
1) get soy sauce, soymilk, the tomato-sauce of 50ml, 12, the centrifugal 15-20min of 000rpm abandons supernatant, keeps precipitation;
2) get 0.5ml bromohexadecane base TMA (TriMethylAmine) and extract the damping fluid dissolution precipitation, it is 2% bromohexadecane base TMA (TriMethylAmine) that bromohexadecane base TMA (TriMethylAmine) is extracted damping fluid, 50mmol/L Tris alkali, pH8.0, the 10mmol/L ethylenediamine tetraacetic acid (EDTA), pH8.0,0.8mol/L sodium-chlor;
3) 65 ℃ of insulation 30～90min, the light and slow frequently therebetween mixing of putting upside down; Add 1: 1 phenol/chloroform of 0.5ml, mixing is to the milkiness shape, and 12, the centrifugal 10-15min of 000rpm is to phase-splitting, draws supernatant liquor with the centrifugal post of crossing of Sephadex-G50 post;
4) dehydrated alcohol that adds isopyknic Virahol or 2.5 times of volumes was placed 1-2 hour in-70 ℃ or-20 ℃, and 12, the centrifugal 10-15min of 000rpm abandons supernatant, keeps precipitation;
5) 70% washing with alcohol precipitation, 12, the centrifugal 10-15min of 000rpm, repeated washing precipitation 1-2 time after the drying at room temperature, adds the Chelex solution of 200 μ l 10% in precipitation, abundant mixing, 100 ℃ are boiled 5-10min;
6) 12, after 000rpm was centrifugal, its supernatant can be directly used in the template of polymerase chain reaction.
2. according to claim 1 a kind of from soy sauce the method for separating residual DNA.It is characterized in that, the preparation method of said Sephadex-G 50 posts is: get 10g Sephadex-G 50 and add repetitive scrubbing in the aseptic distilled water, remove the dextran of solubility, with TE damping fluid balanced gel, autoclaving 15min, the TE damping fluid is a 10mmo/L Tris alkali, 1mmol/L ethylenediamine tetraacetic acid (EDTA), pH7.6; The millipore filtration of 0.45 μ m is put into the test tube post, with Sephadex-G 50 dress posts, 1 * TEN damping fluid balance, add gel and fill fully until the test tube post, 1 * TEN damping fluid is a 10mmol/L Tris alkali, the 1mmol/L ethylenediamine tetraacetic acid (EDTA), 100mmol/L sodium-chlor, pH8.0; Behind the centrifugal 4min of 1600g, continue to add gel and make column volume reach 0.9ml; 0.1ml1 * TEN damping fluid is added in the post, the centrifugal 4min of 1600g, and repeat 2 times, it is standby to fill it up with 4 ℃ of preservations of 1 * TEN damping fluid sealing.
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| CN107365767A (en) * | 2017-09-15 | 2017-11-21 | 广东美格基因科技有限公司 | A kind of method that DNA is extracted from soy sauce |
| CN109880823A (en) * | 2019-04-04 | 2019-06-14 | 中国计量大学 | The extracting method of Walnut Milk DNA |
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| CN109880823A (en) * | 2019-04-04 | 2019-06-14 | 中国计量大学 | The extracting method of Walnut Milk DNA |
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