CN105087550A - Method for rapid and high-flux extraction of plant genome DNA and application of plant genome DNA - Google Patents
Method for rapid and high-flux extraction of plant genome DNA and application of plant genome DNA Download PDFInfo
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- CN105087550A CN105087550A CN201510599233.2A CN201510599233A CN105087550A CN 105087550 A CN105087550 A CN 105087550A CN 201510599233 A CN201510599233 A CN 201510599233A CN 105087550 A CN105087550 A CN 105087550A
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Abstract
The invention belongs to the technical field of molecular biology, particularly relates to a method for rapid and high-flux extraction of plant genome DNA applicable to PCR amplification and further discloses the application of the plant genome DNA. According to the method, through carefully proportioning an SDS extracting solution, the dosage of certain reagents is reduced, fewer reagents are required, and expensive enzymes and other biochemical reagents are not required to be used. The method for extraction of DNA, provided by the invention, is low in cost and simple to operate as a whole, the extraction flux of DNA is high and the time needed is short. The obtained genome DNA can be widely applied to population genetics, molecular marker-assisted breeding, transgenic plant screening and other experiments, which require large-scale genome extraction.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of fast, high-throughput extracts the extracting method being applicable to the plant genome DNA of pcr amplification, and disclose it further and apply.
Background technology
Genomic dna, as the carrier of genetic information and the most basic genetic material, at the important role such as heritable variation, metabolic regulation, is molecular biology and engineered main study subject.The template that plant genome DNA is used as PCR reaction carries out the researchs such as gene clone, location, molecular mark, transgenic line detection.No matter be the structure and function studying DNA of plants, still carry out the conversion of foreign DNA, the research of transduction, the DNA extracting the high molecular of native state exactly from plant tissue that first will do.Therefore, the high-quality DNA extracting q.s is the prerequisite and the necessary guarantee that obtain accurate experimental result.
The method that plant genome DNA extracts has a lot, and the extraction of genomic dna is generally used for building genomic library, Southern hybridizes (comprising RFLP) and PCR isolated genes etc.The genome DNA extracting method of different plant is different; The different tissues of different sorts or one species is because of its cellularstructure and composition difference, and separation method is also variant.
Conventional Plant Genome extracting method mainly comprises SDS method and CTAB method two kinds, but not only has very high nuclease in vegetable cell, and its cell walls contains a large amount of polysaccharide, and its composition is different because species are different.These polysaccharide cause impact to the co-precipitation step in the leaching process of plant genome DNA, reduce the purity of nucleic acid.Make these two kinds of traditional method for extracting processes loaded down with trivial details, take time and effort, tediously long leaching process not only increase genomic dna contaminated may, have impact on its purity, also may reduce activity, lose its natural characteristic; Simultaneously owing to using multiple poisonous and hazardous organic solvent frequently, produce a large amount of toxic waste liquids, therefore abandoned by most of laboratory.
According to the difference of nucleic acid molecular weight or the separation that utilizes specific membranes to realize DNA the specific adsorption of DNA thereupon the RNA isolation kit risen; it appear at the burden alleviating scientific research personnel to a certain extent; be applicable to the extraction of a small amount of high-purity vegetable genomic dna; but its operating process is also too loaded down with trivial details; and expensive, be not suitable for the experiment that population genetics, molecular mark, transfer-gen plant screening etc. need extensive genome to extract.Therefore, up to now, the technological method that quick, easy plant genome DNA extracts also is not formed.
Chinese patent CN102839167A discloses a kind of method that simple and fast high-throughput extracts plant genome DNA, the method is by optimizing the formula of DNA extraction liquid, utilize the Self-made reagent formula of three (methylol) aminomethane hydrochloride Tris-HCl (pH8.0), ethylenediamine tetraacetic acid (EDTA) (EDTA) and Repone K, when not using enzyme reagent and extraction without the need to completing DNA group when liquid nitrogen grinding, not only simple to operate and cost is low.Once can high-throughput process 384 samples, and leaching process only needs 2.5 hours, consuming time short.The DNA that carries only needs 0.5 microlitre to be pcr template, the high and cleanliness without any pollution of purity.The method can be widely used in the researchs such as plant population's genetics, phyletic evolution, molecular mark and degree of depth order-checking, is suitable for industrialization and produces and common biological laboratory science research.But its leaching process is still comparatively loaded down with trivial details, also still there is extraction time longer technical problem.
Summary of the invention
Technical problem to be solved by this invention is the technical problem that the method leaching process extracting plant genome DNA in prior art is loaded down with trivial details, take time and effort, and then provide a kind of can fast, high-throughput extracts and is applicable to the extracting method of the plant genome DNA of pcr amplification, and disclose it further and apply.
For solving the problems of the technologies described above, of the present invention fast, high-throughput extracts the method for plant genome DNA, comprise the steps:
(1) get plant tissue to be extracted and add the mixing of SDS extracting solution, and fully grind; Described SDS extract recipe is: 80-120mMTrisHCl, 40-60mMEDTA, 80-120mMNaCl, 0.5-1.5%w/vSDS, pH8.0;
(2) sample after grinding is heated 5 ~ 10min in 60-70 DEG C, and high speed centrifugation process under room temperature; Pipette samples supernatant liquor subsequently, and add the mixing of isopyknic Virahol, left at room temperature process;
(3) by the high speed centrifugation process again of the sample after standing process, and remove supernatant liquor, add the ethanol that mass concentration is not less than 70% subsequently, fully mix;
(4) by the high speed centrifugation process again of mixed sample, after complete abandoning supernatant, by described sample drying process;
(5) in dried sample, add distilled water, leave standstill at 35-40 DEG C of temperature, and routine preservation, obtain required plant genome DNA and carry out follow-up PCR reaction as template.
Preferably, in described step (1), described SDS extract recipe is: 100mMTrisHCl, 50mMEDTA, 100mMNaCl, 1%w/vSDS, pH8.0.
In described step (1), the quality of described plant tissue and the volume ratio of described SDS extracting solution are 1: 0.2-0.5, and wherein, the proportional units of described quality and volume is g/mL.
Preferably, the quality of described plant tissue and the volume ratio of described SDS extracting solution are 1: 0.3.
In described step (2), (3) and (4), the rotating speed of described high speed centrifugation step is independent of each other is 10000-12000rpm, and centrifugal treating 8-15min.
In described step (1), described plant tissue comprises the seedling of plant, cotyledon, true leaf, tender stem or tender root bit organization.
Described plant comprises cotton, rape, paddy rice, corn, wheat or soybean.
In described step (1), described grinding steps utilizes high-throughput tissue grinder to grind.
In described step (4), described drying step is vacuum-drying.
In described step (4), described drying temperature is 50-70 DEG C.
Method that is quick, high-throughput extraction plant genome DNA of the present invention passes through the meticulous proportioning of SDS extracting solution, (concentration of SDS is down to 1% by 2% of routine to reduce the usage quantity of portion of reagent, the concentration of NaCl is down to 100mM by the 500mM in traditional extraction liquid), not only required reagent is few, and without the need to using expensive enzyme and other biochemical reagents (in prior art, other extracting method also needs to use CTAB, mercaptoethanol etc.).Make the method for extraction DNA of the present invention not only with low cost like this, average each sample cost 0.2 ~ 0.5 yuan (other ordinary method needs 1 ~ 9 yuan); And whole method operation is comparatively simple, only need 5 steps can complete (other ordinary method needs 10 ~ 20 steps); The most important thing is, the extraction flux of described DNA is high, required time is short, and disposable operation 192 whole experimentations of sample about need 1h ~ 1.5h (other ordinary method about needs 3 ~ 6h).The method of the invention is applicable to various plants (cotton, rape, paddy rice, corn, wheat, soybean etc.) simultaneously; higher compared to other ordinary method suitabilities, gained genomic dna can be widely used in the experiment that the extensive genome of the needs such as population genetics, molecular mark, transfer-gen plant screening extracts.
Accompanying drawing explanation
In order to make content of the present invention be more likely to be clearly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein,
Fig. 1 is the plant genome DNA electrophoretogram utilizing each sample of the present invention and full formula gold Plant Genome to extract test kit extraction; Wherein, sequence number 1-6 be respectively utilize the present invention to extract cotton, corn, rape, paddy rice, soybean, Wheat volatiles DNA electrophoretogram, 1 '-6 ' be respectively utilize full formula gold Plant Genome extract test kit extract cotton, corn, rape, paddy rice, soybean, Wheat volatiles DNA electrophoretogram;
Fig. 2 is with each sample Plant Genome of the present invention's extraction for template, the electrophoretogram of each plant internal standard gene that increases.
Embodiment
Embodiment 1
Clip fresh corn blade is about 1g, and blade can be directly used in genome and extract, and also can save backup at 4 DEG C, extracts in 10 days.The steel ball that blade and diameter are about 3mm is put into 1.5ml centrifuge tube, adds the SDS extracting solution that 300 μ L improve.Described SDS extract recipe is: 100mMTrisHCl, 50mMEDTA, 100mMNaCl, 1%w/vSDS, pH8.0.
Moved to by centrifuge tube in high-throughput tissue grinder sample milling machine, arranging frequency is 33 times/second, concussion 30s, and the sample after grinding is put into 65 DEG C of heating 5 ~ 10min, period puts upside down mixing 3 ~ 4 times.Centrifuge tube after heating is put into whizzer, under 12000rpm condition, the centrifugal 10min of room temperature.In every pipe, draw 100 μ L supernatant liquors, put into 96 orifice plates, add isopyknic Virahol with the volley of rifle fire, every plate covers silicagel pad, and mixing of turning upside down, room temperature leaves standstill 30min.
By the sample recentrifuge 10min under 12000rpm condition after leaving standstill, silicagel pad opened, get rid of clean supernatant, then in 96 orifice plates, to add 200 μ L mass concentrations with the volley of rifle fire be the ethanol of 70%, turn upside down 96 orifice plates, and flick tube wall.
By described sample centrifugal 10min under 12000rpm condition again, silicagel pad is opened, gets rid of clean supernatant.96 orifice plates are inverted in 1min on clean thieving paper, and then being faced up by 96 orifice plates is placed on 60 DEG C of dry 2min in vacuum-drying instrument.
In every hole of described 96 orifice plates, add 40 μ L distilled waters, refrigerator routine preservation is for subsequent use or-20 DEG C of conventional cryopreservation are for subsequent use to put into 4 DEG C after 37 DEG C of placement 1h, carries out follow-up PCR reaction as template.
Genome extracts and extracts test kit in contrast with full formula gold Plant Genome.Get 1 ~ 2 μ L as template, amplification corn internal standard gene zSSIIb (primer and reaction conditions are pressed No. 869, Ministry of Agriculture bulletin-3-2007 and performed).
Embodiment 2
Clip fresh soyabean blade is about 1g, and blade can be directly used in genome and extract, and also can save backup at 4 DEG C, extracts in 10 days.The steel ball that blade and diameter are about 3mm is put into 1.5ml centrifuge tube, adds the SDS extracting solution that 200 μ L improve.Described SDS extract recipe is: 80mMTrisHCl, 60mMEDTA, 120mMNaCl, 0.5%w/vSDS, pH8.0.
Moved to by centrifuge tube in high-throughput tissue grinder sample milling machine, arranging frequency is 33 times/second, concussion 30s, and the sample after grinding is put into 60 DEG C of heating 5 ~ 10min, period puts upside down mixing 3 ~ 4 times.Centrifuge tube after heating is put into whizzer, under 10000rpm condition, the centrifugal 10min of room temperature.In every pipe, draw 100 μ L supernatant liquors, put into 96 orifice plates, add isopyknic Virahol with the volley of rifle fire, every plate covers silicagel pad, and mixing of turning upside down, room temperature leaves standstill 30min.
By the sample recentrifuge 10min under 12000rpm condition after leaving standstill, silicagel pad opened, get rid of clean supernatant, then in 96 orifice plates, to add 200 μ L mass concentrations with the volley of rifle fire be the ethanol of 65%, turn upside down 96 orifice plates, and flick tube wall.
By described sample centrifugal 10min under 12000rpm condition again, silicagel pad is opened, gets rid of clean supernatant.96 orifice plates are inverted in 1min on clean thieving paper, and then being faced up by 96 orifice plates is placed on 70 DEG C of dry 2min in vacuum-drying instrument.
In every hole of described 96 orifice plates, add 40 μ L distilled waters, refrigerator routine preservation is for subsequent use or-20 DEG C of conventional cryopreservation are for subsequent use to put into 4 DEG C after 37 DEG C of placement 1h, carries out follow-up PCR reaction as template.
Genome extracts and extracts test kit in contrast with full formula gold Plant Genome.Get 1 ~ 2 μ L as template, amplification soybean internal standard gene Lectin (primer and reaction conditions are pressed agricultural industry criteria NY-T675-2003 and performed).
Embodiment 3
The fresh rice leaf of clip is about 1g, and blade can be directly used in genome and extract, and also can save backup at 4 DEG C, extracts in 10 days.The steel ball that blade and diameter are about 3mm is put into 1.5ml centrifuge tube, adds the SDS extracting solution that 500 μ L improve.Described SDS extract recipe is: 120mMTrisHCl, 40mMEDTA, 80mMNaCl, 1.5%w/vSDS, pH8.0.
Moved to by centrifuge tube in high-throughput tissue grinder sample milling machine, arranging frequency is 33 times/second, concussion 30s, and the sample after grinding is put into 70 DEG C of heating 5 ~ 10min, period puts upside down mixing 3 ~ 4 times.Centrifuge tube after heating is put into whizzer, under 12000rpm condition, the centrifugal 10min of room temperature.In every pipe, draw 100 μ L supernatant liquors, put into 96 orifice plates, add isopyknic Virahol with the volley of rifle fire, every plate covers silicagel pad, and mixing of turning upside down, room temperature leaves standstill 30min.
By the sample recentrifuge 10min under 12000rpm condition after leaving standstill, silicagel pad opened, get rid of clean supernatant, then in 96 orifice plates, to add 200 μ L mass concentrations with the volley of rifle fire be the ethanol of 75%, turn upside down 96 orifice plates, and flick tube wall.
By described sample centrifugal 10min under 12000rpm condition again, silicagel pad is opened, gets rid of clean supernatant.96 orifice plates are inverted in 1min on clean thieving paper, and then being faced up by 96 orifice plates is placed on 50 DEG C of dry 2min in vacuum-drying instrument.
In every hole of described 96 orifice plates, add 40 μ L distilled waters, refrigerator routine preservation is for subsequent use or-20 DEG C of conventional cryopreservation are for subsequent use to put into 4 DEG C after 37 DEG C of placement 1h, carries out follow-up PCR reaction as template.
Genome extracts and extracts test kit in contrast with full formula gold Plant Genome.Get 1 ~ 2 μ L as template, amplifying rice internal standard gene SPS (primer and reaction conditions are pressed No. 1485, Ministry of Agriculture bulletin-5-2010 and performed).
Embodiment 4
The present embodiment carries out DNA extraction to Cotton tissue, and whole extraction step is identical with embodiment 1.
Embodiment 5
The present embodiment carries out DNA extraction to rapeseed plant tissue, and whole extraction step is identical with embodiment 1.
Embodiment 6
The present embodiment carries out DNA extraction to wheat plant tissue, and whole extraction step is identical with embodiment 1.
Experimental example
To extracting the cotton obtained in the above embodiment of the present invention, corn, rape, paddy rice, soybean, Wheat volatiles DNA carries out cataphoretic determination, fig. 1 illustrates utilize the present invention to extract plant genome DNA and adopt full formula gold Plant Genome to extract the electrophoretogram of the plant genome DNA (in contrast) that test kit extracts, wherein, sequence number 1-6 is respectively the cotton utilizing the present invention to extract, corn, rape, paddy rice, soybean, Wheat volatiles DNA electrophoretogram, 1 '-6 ' cotton utilizing full formula gold Plant Genome to extract test kit extraction is respectively, corn, rape, paddy rice, soybean, Wheat volatiles DNA electrophoretogram (marker is trans15KDNAmarker), following table 1 gives the Comparative result that the concentration of the DNA group utilizing spectrophotometric determination the present invention to extract and quality and full formula gold reagent box measure.
Concentration and the quality of DNA are extracted in this experiment of table 1 spectrophotometric determination
According to table 1 data presentation, according to spectrophotometric measurement, the concentration provided plant utilization the present invention being extracted to DNA is obviously greater than the concentration utilizing test kit to extract DNA, and the purity of DNA carried not as good as kit method.On the whole, its A of DNA of the present invention's extraction
260/280all lower than 1.8, illustrate containing more protein in DNA, and its A
260/230major part illustrates in DNA containing more polysaccharide or polyphenol lower than 2.0.But the corn utilizing the present invention to extract and the genome concentration of soybean are far above RNA isolation kit, purity is suitable with RNA isolation kit.
And Fig. 2 gives with the Plant Genome of the present invention's extraction for template, the electrophoretogram of each plant internal standard gene that increases.The plant genome DNA that the visible the present invention of utilization extracts, as template, effectively can amplify the internal standard gene of each plant.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (10)
1. quick, high-throughput extracts a method for plant genome DNA, it is characterized in that, comprises the steps:
(1) get plant tissue to be extracted and add the mixing of SDS extracting solution, and fully grind; Described SDS extract recipe is: 80-120mMTrisHCl, 40-60mMEDTA, 80-120mMNaCl, 0.5-1.5%w/vSDS, pH8.0;
(2) sample after grinding is heated 5 ~ 10min in 60-70 DEG C, and high speed centrifugation process under room temperature; Pipette samples supernatant liquor subsequently, and add the mixing of isopyknic Virahol, left at room temperature process;
(3) by the high speed centrifugation process again of the sample after standing process, and remove supernatant liquor, add the ethanol that mass concentration is not less than 70% subsequently, fully mix;
(4) by the high speed centrifugation process again of mixed sample, after complete abandoning supernatant, by described sample drying process;
(5) in dried sample, add distilled water, leave standstill at 35-40 DEG C of temperature, and routine preservation, obtain required plant genome DNA and carry out follow-up PCR reaction as template.
2. according to claim 1 fast, high-throughput extracts the method for plant genome DNA, it is characterized in that, in described step (1), described SDS extract recipe is: 100mMTrisHCl, 50mMEDTA, 100mMNaCl, 1%w/vSDS, pH8.0.
3. according to claim 1 and 2 fast, high-throughput extracts the method for plant genome DNA, it is characterized in that, in described step (1), the quality of described plant tissue and the volume ratio of described SDS extracting solution are 1: 0.2-0.5, wherein, the proportional units of described quality and volume is g/mL.
4. according to claim 3 fast, high-throughput extracts the method for plant genome DNA, it is characterized in that, the quality of described plant tissue and the volume ratio of described SDS extracting solution are 1: 0.3.
5. the method for plant genome DNA is extracted according to claim 1-4 arbitrary described quick, high-throughput, it is characterized in that, in described step (2), (3) and (4), the rotating speed of described high speed centrifugation step is independent of each other is 10000-12000rpm, and centrifugal treating 8-15min.
6. extract the method for plant genome DNA according to claim 1-5 arbitrary described quick, high-throughput, it is characterized in that, in described step (1), described plant tissue comprises the seedling of plant, cotyledon, true leaf, tender stem or tender root bit organization.
7. according to claim 6 fast, high-throughput extracts the method for plant genome DNA, it is characterized in that, described plant comprises cotton, rape, paddy rice, corn, wheat or soybean.
8. extract the method for plant genome DNA according to claim 1-7 arbitrary described quick, high-throughput, it is characterized in that, in described step (1), described grinding steps utilizes high-throughput tissue grinder to grind.
9. extract the method for plant genome DNA according to claim 1-8 arbitrary described quick, high-throughput, it is characterized in that, in described step (4), described drying step is vacuum-drying.
10. according to claim 9 fast, high-throughput extracts the method for plant genome DNA, it is characterized in that, in described step (4), described drying temperature is 50-70 DEG C.
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| CN112779246A (en) * | 2020-12-30 | 2021-05-11 | 浙江大学 | Plant genome DNA extracting solution and plant genome DNA extracting method |
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Application publication date: 20151125 |