CN102206630B - Method and kit for extracting total DNA of soil and sediment - Google Patents
Method and kit for extracting total DNA of soil and sediment Download PDFInfo
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Abstract
The invention relates to a method and a kit for extracting total DNA of soil and sediment. The method comprises the following steps of: dissolving and removing humic acid from the soil and sediment to the greatest extent by using a humic acid dissolving solution; performing complex-precipitation on the residual humic acid; randomly cracking and releasing genomic DNA in bacteria; centrifuging to remove complexed humic acid and protein from the solution; and regulating supernate containing DNA by using an acid-base regulating agent and a high-concentration salt solution, and specifically adsorbing by using a silica gel adsorption film to make the genomic DNA purified and recovered. By the method, a plurality of samples can be extracted in short time, the normal operating time is less than 70 minutes, and massive DNA samples having high purity and suitable for downstream analysis can be obtained. By the kit established according to the method, the obtained DNA samples have high purity without degradation and can be directly used for subsequent molecular biology analysis; meanwhile, a step of purifying the DNA by toxic reagents such as phenol and chloroform in the traditional DNA extraction method is avoided, and the invention has wide application prospect.
Description
Technical field
The invention belongs to soil microbe genome DNA separating and purifying technology field, be specifically related to a kind of soils and sediments total DNA extraction method and extract test kit.
Background technology
The molecular fingerprint graphical spectrum technology has become the microbial ecological of generally using in the fields such as microbial ecology, environmental, environmental engineering and has learned a skill, yet the extractive technique of (such as soil, ocean and lake sediment etc.) total DNA gets more and more people's extensive concerning as committed step and the core content of this technical system in the environmental sample.The specificity of total DNA extraction, concentration, purity etc. directly have influence on the objective reality of biological community structure in the soils and sediments, are one of the more contents of disputing in the Abroad in Recent Years periodical refereeing procedure.
At present, for from soils and sediments, obtaining total DNA, still do not have the preferred approach of consistent approval, and generally the enzymatic lysis method of application, pharmaceutical chemicals extraction method etc. can only work for the sensitive organism in the sample, and can't remove the humic acid in the sample.These methods all artificial change real biological community structure in the environment, and have following problem: 1) the DNA extraction process is numerous and diverse, overlong time, and need more large-scale instrument and equipment needs long water-bath process such as enzyme extraction method; 2) the general dna leaching process is often used the chemical reagent of high malicious high pollution, as using " three cause " reagent such as phenol/chloroform in the DNA purge process; 3) DNA that extracts is second-rate, and fragment is more, has the pollutions such as humic acid, RNA, protein; 4) amount of DNA is especially obtained DNA very little from settling, can't satisfy the needs of downstream experiment.Therefore, set up a kind of method of effectively from soils and sediments, extracting total DNA and become the task of top priority.
Summary of the invention
The purpose of this invention is to provide a kind of method that from soils and sediments, obtains fast total DNA, and provide complete test kit product innovation, use this test kit, the investigator can obtain the DNA of needs fast from environmental sample, to carry out follow-up molecular biology research.
The present invention at first uses the humic acid lysate, farthest dissolve and remove humic acid in the soils and sediments, to remain again humic acid and carry out complex-precipitation, then without the DNA that selectively discharges in all bacterial bodies, complexing humic acid and protein in the centrifugal rear removal solution, contain the supernatant solution of DNA then by after the high level salt solution adjusting, be able to purifying with the special absorption of silica gel adsorption film and reclaim.
Soils and sediments total DNA extraction method of the present invention in turn includes the following steps:
1) removes the most of humic acid of sample with the humic acid lysate, be precipitated;
2) to above-mentioned steps 1) deposit sample in add the humic acid complexing agent, having suspended to precipitate makes the abundant complexing of remaining humic acid, centrifugal humic acid complex compound and the bacteria samples of making fully precipitates;
3) to above-mentioned steps 2) precipitation in add cell pyrolysis liquid, vortex oscillation discharges bacteria total DNA, obtains containing the supernatant liquor of bacteria total DNA after centrifugal;
4) to above-mentioned steps 3) supernatant liquor in add acid-base modifier, the centrifugal supernatant solution that obtains removing protein and sugar class material;
5) to above-mentioned steps 4) supernatant solution in add high salt solvent after, cross silica gel adsorption film adsorption of DNA;
6) with above-mentioned steps 5) after the DNA of absorption cleans, obtain total DNA of sample with the elutriant wash-out.
Above-mentioned humic acid complexing agent is the calcium ion aqueous solution, is preferably calcium chloride solution, is 0.20mol/L, and pH 5.5～7.5; And the weight in wet base of soil or sediment sample is 1.5g/mmol/L with the adding proportion of calcium ion.
Above-mentioned acid-base modifier is 0.5～1.5mol/L Potassium ethanoate, and pH 4.8～5.0, has the regulator solution system to acid, with further precipitation humic acid and glucide, and the effect of denatured protein.
Above-mentioned step 3) vortex oscillation time of releasing is 10～12min.
Extraction test kit according to above-mentioned extracting method is set up comprises following component:
1) humic acid lysate and extra large sand, wherein extra large sand diameter is 1～2mm, the mass volume ratio of extra large sand and humic acid lysate is 0.2～0.3g/mL;
2) humic acid complexing agent: the 0.20mol/L calcium chloride solution, pH 5.5～7.5
3) cell pyrolysis liquid: 50～100mmol/L Tris-HCl, 1.5～3.5mol/L NaCl, 1%～3%CTAB, pH 8.0～10.0;
4) denaturing agent: 20%SDS, pH 9.0;
5) acid-base modifier: 0.5～1.5mol/L Potassium ethanoate, pH 4.8～5.0;
6) high salt solvent: 3.0～6.0mol/L Guanidinium hydrochloride, pH 5.0～8.0;
7) scavenging solution: 70% ethanol;
8) elutriant: 2～10mmol/L Tris-HCl, pH 8.0～9.0;
9) genomic dna adsorption column: the 2mL centrifuge tube suit that comprises silica gel adsorption film inner prop.
The test kit of setting up according to method of the present invention can extract a plurality of samples simultaneously at 70min in the time, the DNA sample purity that obtains is high, without degraded, can be directly used in follow-up molecular biological analysis, avoided simultaneously traditional DNA extraction method to need the step of the toxic agent purify DNAs such as phenol/chloroform, had broad application prospects.
Description of drawings
Fig. 1: use the different soils sample DNA electrophoresis result figure that test kit of the present invention extracts.
Fig. 2: different soils sample DNA 16S rRNA gene PCR amplification electrophoresis result.
Fig. 3: different soils sample DNA 16S rRNA gene V3 district pcr amplification electrophoresis result.
Fig. 4: different soils sample DNA ammonia oxidation bacteria distinguished sequence pcr amplification electrophoresis result.
Wherein, M is marker, and each fragment marks in figure right; 1,2,3 and 4 are respectively farmland soil, meadow soil, native, the oceanic sediment of wetland, the negative contrast of C.
Embodiment
Below in conjunction with specific embodiment method of the present invention is described in detail.
The present invention obtains the method for total DNA from soils and sediments, in turn include the following steps:
1) process sample with extra large sand and humic acid lysate, the centrifugal deposit sample that obtains removing most of humic acid:
Get 0.3g diameter 1～2mm sea sand in the centrifuge tube of 2mL, carry out high pressure steam sterilization, add humic acid lysate (100～200mmol/L Tris, the 50～100mmol/LNa of 1.0～1.5mL sterilization
4P
2O
7, 50～100mmol/L Na
2EDTA, 1.0～3.0%PVP, 100～200mmol/L NaCl, 0.05～0.1%Triton X-100, pH 8.0～10.0), place room temperature for subsequent use;
Soil or sediment sample 0.3g are joined in the centrifuge tube that humic acid lysate and extra large sand are housed middling speed vortex vibration 1～3min, fully mixing, humic acid in the pedotheque is fully discharged, then the centrifugal 30s of 10000 * g inhales as far as possible and abandons supernatant, is precipitated sample;
2) to above-mentioned steps 1) deposit sample in add the humic acid complexing agent, having suspended to precipitate makes the abundant complexing of remaining humic acid, centrifugal extra large sand, humic acid complex compound and the bacteria samples of making fully precipitates:
To above-mentioned steps 1) deposit sample add 1mL 0.20mol/L calcium chloride solution, pH 5.5～7.5, middling speed vortex 1～3min then, complete mixing, the centrifugal 30s of 10000 * g inhales as far as possible and abandons supernatant, obtains containing the precipitation of DNA.
Calcium ion can with electronegative larger molecular organics complexing, form infusible precipitate, humic acid, protein and DNA etc. are electronegative larger molecular organics under neutrallty condition, but this moment, bacterial cell was not yet broken, DNA does not also discharge, so the calcium ion that adds only forms infusible precipitate with the humic acid complexing.And other humic acid complexometric reagent owing to can not form stable complex compound sediment with humic acid, can make again humic acid enter in the sample system in follow-up lysis.The present invention need to select the calcium salt of good water solubility, preferably calcium chloride solution.
In addition, the calcium ion concn in the humic acid complexing agent of adding also produces important impact to DNA extraction.Calcium ion concn is excessively low, will make in the sample humic acid complexing incomplete, and too high with remaining too much, affect output and the quality of follow-up DNA.Originally studies show that, in step 1) in, get under 0.3g soil or the sedimental condition, add 1mL 0.2mol/L calcium chloride solution (ratio that is sample weight in wet base and calcium ion is 1.5g/mmol/L) and just can will remain the complete complexing of humic acid, and less to the DNA yield effect.
3) to above-mentioned steps 2) precipitation in add cell pyrolysis liquid and denaturing agent and vortex oscillation and discharge bacteria total DNA, obtain containing the supernatant liquor of bacteria total DNA after centrifugal:
Add 720 μ L cell pyrolysis liquids, 50～100mmol/L Tris-HCl in the precipitation, 1.5～3.5mol/LNaCl, 1%～3%CTAB, pH 8.0～10.0; With 80 μ L denaturing agent 20%SDS, pH 9.0, top speed vortex oscillation 10～12min, and the centrifugal 60s of 10000 * g transfers to supernatant liquor (＜650 μ L avoid drawing the upper strata bubble) in the new pipe;
4) to above-mentioned steps 3) supernatant liquor in add acid-base modifier, the centrifugal supernatant solution that obtains removing protein and sugar class material:
Add 100 μ L acid-base modifiers, 0.5～1.5mol/L Potassium ethanoate, pH 4.8～6.0; Put upside down mixing 5 times, 4 ℃ leave standstill 5min, the centrifugal 60s of 10000 * g, and supernatant (＜700 μ L) moves to new pipe;
Step 3) supernatant liquor that obtains in is less than 650 μ L, and the acid-base modifier of adding is 100 μ L, is to obtain comparatively ideal hydrochlorate regulating effect, and the volume ratio of the two needs greater than 6.5.
5) to above-mentioned steps 4) supernatant solution in add high salt solvent after, cross silica gel adsorption film adsorption of DNA:
To step 4) supernatant solution in add the high salt solvent of 1400 μ L: 3.0～6.0mol/L Guanidinium hydrochloride, pH5.0～8.0; Behind the mixing, minute three times (getting 700 μ L) adds same silica gel adsorption film adsorption of DNA, the centrifugal 30s of 10000 * g at every turn;
To above-mentioned steps 4) in the supernatant solution that obtains, when the salts solution that adds high density is competed available water with dna molecular, dna molecular will be separated out and silica gel adsorption film in organic phase is combined.Guanidinium hydrochloride has the characteristic of salt, can high density be dissolved in water simultaneously, and this is that other salts are incomparable, and this also is the reason of applying high density Guanidinium hydrochloride of the present invention.
6) with above-mentioned steps 5) after the DNA of absorption cleans, obtain total DNA of sample with the elutriant wash-out:
Add 700 μ L70% ethanol, the centrifugal 60s of 10000 * g, after removing filtered solution, once centrifugal again, adsorption column is placed new pipe, then use 100 μ L elutriants: 2～10mmol/L Tris-HCl, pH 8.0～9.0 joins in the adsorption column, in 60 ℃ of water-bath 5min, and the centrifugal 60s of 10000 * g, collect filtered solution, in-20 ℃ of preservations.
Extraction test kit of the present invention comprises following component:
1) humic acid lysate and extra large sand, wherein extra large sand diameter is 1～2mm, the mass volume ratio of extra large sand and humic acid lysate is 0.2～0.3g/ml;
2) humic acid complexing agent: the 0.20mol/L calcium chloride solution, pH 5.5～7.5;
3) cell pyrolysis liquid: 50～100mmol/L Tris-HCl, 1.5～3.5mol/LNaCl, 1%～3%CTAB, pH 8.0～10.0;
4) denaturing agent: 20%SDS, pH 9.0;
5) acid-base modifier: 0.5～1.5mol/L Potassium ethanoate, pH 4.8～5.0;
6) high salt solvent: 3.0～6.0mol/L Guanidinium hydrochloride, pH 5.0～8.0;
7) scavenging solution: 70% ethanol;
8) elutriant: 2～10mmol/L Tris-HCl, pH 8.0～9.0;
9) genomic dna adsorption column: the 2ml centrifuge tube suit that comprises silica gel adsorption film inner prop.
Use this test kit, the simple device that only needs the laboratories such as the general microbiology such as supercentrifuge, vortex oscillation device, water-bath and micropipet, molecular biology of conventional non-frozen type all to possess, for skilled investigator, extracting simultaneously 6 sample DNA times can foreshorten within the 70min, saved greatly the time of researcher preciousness, simultaneously can obtain high-quality DNA sample, the molecular biology operation unrestraint to the downstream shows great application potential.By to farmland soil, the meadow is native, wetland is native, the extraction test of total DNA in the oceanic sediment, all obtains desirable result, and does not affect the follow-up molecular biological analysis such as pcr amplification.
Embodiment 1: the selection of humic acid complexing agent concentration
Use this test kit, take wetland soil, oceanic sediment and farmland soil as object, inquire into the humic acid complexing agent concentration to the impact of extraction efficiency.Sample size is 0.3g, and wherein calcium chloride solution concentration is respectively 0,0.1,0.2,0.3,0.4,0.5mol/L, and each adds 1ml, extracts about 60min of time spent according to method of the present invention.The DNA that extracts is carried out electrophoresis, dyeing, photograph, and use ultraviolet spectrophotometer and carry out qualitative and quantitative analysis.The result shows, calcium chloride solution concentration is respectively 0 and (the ratio of sample weight in wet base and calcium ion 〉=3g/mmol) during 0.1mol/L, although the rate of recovery of DNA is higher, be 10～15 μ g/g dry ground, but the dna solution that extracts is yellow, contains a large amount of humic acid, shows that humic acid is not completely removed, the calcium chloride relative quantity that adds is excessively low, and such DNA sample will have to the enzyme in the molecular biology experiment great restraining effect.Equally, using concentration and be 0.3mol/L above (is that the calcium chloride solution of the ratio≤1g/mmol/L) of sample weight in wet base and calcium ion is when being the humic acid complexing agent, although dna solution is transparent limpid, but the DNA rate of recovery is excessively low, be lower than the detection limit value during electrophoresis, ultraviolet detection shows and is lower than the 100ng/g dry ground that this shows that too high calcium chloride solution is residual too many, also complexing a large amount of dna moleculars, such DNA sample can't satisfy follow-up molecular biology needs.And when adding 1mL 0.2mol/L calcium chloride solution (ratio that is sample and calcium ion is 1.5g/mmol/L), the DNA concentration of not only extracting high (5～7 μ g/g), and quality can satisfy the follow-up molecular biological requirements such as library construction, denaturing gradient gel electrophoresis (DGGE).
Embodiment 2: bacteria cell cracking vortex oscillation selection of time
Use this test kit, native as object take 0.3g wetland soil, farmland respectively, the vortex oscillation time that adds after cell pyrolysis liquid and the denaturing agent is optimized.Wherein the humic acid complexing agent is with the calcium chloride solution of the 0.2mol/L of 1ml, and pH 7.5, and the vortex oscillation time during lysis is selected respectively 5min, 10min, and 15min extracts according to method of the present invention, about 70min of time spent.DNA electrophoresis, dyeing, photograph, and use ultraviolet spectrophotometer and carry out qualitative and quantitative analysis.The result shows that the environmental sample DNA rate of recovery of vibration 5min is obviously on the low side, is lower than 1.0 μ g/g dry ground; And after being higher than 15min, the DNA rate of recovery there is no obvious increase, and a large amount of DNA fragments occurred, and this is unallowed for the molecular biology experiment that needs Long fragment gene group DNA, is advisable so duration of oscillation is chosen 10～12min.
Embodiment 3: extract total DNA from different pedotheques
The extraction conditions of using this test kit and determining, respectively to farmland soil, the meadow is native, wetland is native, total DNA extracts test in the oceanic sediment, extracts about 60min of time spent according to method of the present invention.Get 10 μ L DNA and carry out electrophoresis, gel dyes with EB, and gel imaging system is taken a picture, and uses ultraviolet spectrophotometer and carry out qualitative and quantitative analysis, and electrophoresis result as shown in Figure 1.According to this figure, total DNA that this test kit can obtain from different soils and sediments, dna fragmentation length is at 10～20kb, and not degraded does not have RNA to pollute yet.Respectively each sample is carried out uv-absorbing and detect, show that the DNA sample that obtains forms the DNA absorption peak of homogeneous at the 260nm place, do not have obvious albumen absorption peak OD to occur at the 280nm place
260/ OD
280=1.8～1.9, illustrate that the DNA that obtains is purer, there are not albumen and RNA to pollute, calculate and learn that the rate of recovery of DNA is 5～12 μ g/g dry ground.
For confirming that can use this test kit extraction DNA quality satisfy the molecular biology needs, take the DNA that obtains as template, use respectively universal primer 8F/1541R, denaturing gradient gel electrophoresis (DGGE) the 16S rRNA gene V3 district special primer 341F/534R commonly used of bacterial 16 S rRNA full length gene, and the special 16S rRNA gene primer CTO189f/CTO654r of ammonia oxidizing bacteria carries out pcr amplification.After PCR finishes, get 5 μ L electrophoresis, the result is respectively such as Fig. 2, Fig. 3 and Fig. 4.Fig. 2 demonstration obtains the 16S rna gene near total length take 8F/1541R as primer, about 1500bp, and concentration is approximately 10ng/tL.The structure that can be used for 16S rRNA gene library behind this product purification is to resolve kind, the content and component proportion of microorganism in the environment.Fig. 3 shows that each sample has all obtained about 200bp target sequence, and concentration is approximately 20ng/ μ L, can be used for DGGE and analyzes, with the structure of analyzing microbial community and dynamically.After Fig. 4 demonstration was carried out PCR take TO189f/CTO654r as primer, each sample had all obtained the about 500bp target sequence of length, and concentration is approximately 10ng/ μ L.As seen, use this test kit, can from soil, sediment sample, extract high-quality DNA, can satisfy the biological needs of downstream molecules.
Claims (4)
1. soils and sediments total DNA extraction test kit comprises following component:
1) efficient DNA separator column: comprise humic acid lysate and extra large sand; The prescription of described humic acid lysate is 100 ~ 200 mmol/L Tris, 50 ~ 100 mmol/L Na
4P
2O
7, 50 ~ 100 mmol/L Na
2EDTA, 1.0 ~ 3.0% PVP, 100 ~ 200 mmol/L NaCl, 0.05 ~ 0.1% Triton X-100, pH 8.0 ~ 10.0;
2) humic acid complexing agent: 0.20 mol/L calcium chloride solution, pH 5.5 ~ 7.5;
3) cell pyrolysis liquid: 50 ~ 100 mmol/L Tris-HCl, 1.5 ~ 3.5 mol/L NaCl, 1% ~ 3% CTAB, pH 8.0 ~ 10.0;
4) denaturing agent: 20%SDS, pH 9.0;
5) acid-base modifier: 0.5 ~ 1.5 mol/L Potassium ethanoate, pH 4.8 ~ 6.0;
6) high salt solvent: 3.0 ~ 6.0 mol/L Guanidinium hydrochlorides, pH 5.0 ~ 8.0;
7) scavenging solution: 70% ethanol;
8) elutriant: 2 ~ 10 mmol/L Tris-HCl, pH 8.0 ~ 9.0;
9) genomic dna adsorption column: the 2 mL centrifuge tubes suit that comprises silica gel adsorption film inner prop.
2. DNA extraction test kit as claimed in claim 1 is characterized in that the mass volume ratio g/mL of extra large sand and humic acid lysate is 0.2 ~ 0.3 in the described efficient DNA separator column.
3. method of utilizing test kit claimed in claim 1 to extract the total DNA of soils and sediments in turn includes the following steps:
1) removes the most of humic acid of sample with the humic acid lysate, obtain the sample precipitation;
2) in the sample precipitation of above-mentioned step 1), add the humic acid complexing agent, make the abundant complexing of remaining humic acid, centrifugal humic acid complex compound and the bacteria samples precipitation of making;
3) to above-mentioned steps 2) precipitation in add cell pyrolysis liquid, vortex oscillation discharges bacteria total DNA, the centrifugal supernatant solution that obtains;
4) in above-mentioned step 3) supernatant solution, add acid-base modifier, the centrifugal supernatant liquor that obtains; And the volume ratio of the acid-base modifier of supernatant solution and adding is not less than 6.5;
5) in above-mentioned step 4) supernatant liquor, add high salt solvent, cross silica gel adsorption film adsorption of DNA;
6) with above-mentioned steps 5) absorption DNA clean, obtain total DNA of sample with the elutriant wash-out;
Step 2 wherein) the humic acid complexing agent that adds in the sample precipitation is calcium chloride solution;
The high salt solvent of step 5) is 3.0 ~ 6.0 mol/L Guanidinium hydrochlorides, and pH 5.0 ~ 8.0.
4. extracting method as claimed in claim 3 is characterized in that be 10 ~ 12 min vortex oscillation time of releasing of described step 3).
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| CN102382821B (en) * | 2011-11-18 | 2016-02-10 | 重庆邮电大学 | A kind of genome DNA extracting method |
| CN103374566A (en) * | 2012-04-23 | 2013-10-30 | 内蒙古大学 | Efficient method for extracting purified total DNA (deoxyribonucleic acid) from lake sediment sample |
| CN103627703B (en) * | 2013-12-19 | 2017-10-17 | 江西省科学院微生物研究所 | The synchronous Total DNA extraction method and kit for removing humic acid and mycoprotein |
| CN105087547A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Method for extracting bacterial genomes in ores |
| CN110499379A (en) * | 2019-09-18 | 2019-11-26 | 武汉轻工大学 | Primer, the method and kit for detecting Lactobacillus sp. |
| CN112201314B (en) * | 2020-09-18 | 2024-05-03 | 北京望石智慧科技有限公司 | Method and device for extracting molecular fingerprint and calculating correlation based on molecular fingerprint |
| CN114107286A (en) * | 2021-12-06 | 2022-03-01 | 哈尔滨市青蛙生物科技有限责任公司 | Universal soil genome DNA extraction kit and use method thereof |
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| Title |
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| 李靖宇等.湿地土壤微生物DNA提取及其脱腐技术.《微生物学通报》.2010,参见正文第1131页右栏1.2节. * |
| 王丽娜等.三种土壤微生物总DNA提取方法的比较.《基因组学与应用生物学》.2009,正文第332页左栏第1.2节. * |
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