CN109371009A - A kind of method that high throughput maize leaf DNA is extracted - Google Patents
A kind of method that high throughput maize leaf DNA is extracted Download PDFInfo
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- CN109371009A CN109371009A CN201811251487.5A CN201811251487A CN109371009A CN 109371009 A CN109371009 A CN 109371009A CN 201811251487 A CN201811251487 A CN 201811251487A CN 109371009 A CN109371009 A CN 109371009A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The present invention relates to the extracting methods of DNA, specifically disclose a kind of method that high throughput maize leaf DNA is extracted.The method is that pretreatment fluid is added in maize leaf, is ground；Pretreatment fluid is sucked out to discard, DNA extracting solution is added；Water-bath is extracted after mixing, and extracting solution is centrifuged, and takes supernatant, and isopropanol precipitating DNA is added, and is mixed, and centrifugation discards supernatant；Be added alcohol wash precipitating DNA, mix, centrifugation, discard supernatant to get.The method of the invention carries out sample process without using liquid nitrogen, and without using organic solvents such as chloroforms, comparatively safe nontoxic；The present invention is by being added pretreatment fluid, effectively prevent the oxidation of maize leaf during the grinding process, and joined a certain proportion of polyvinylpyrrolidone (PVP) in pretreatment fluid and extracting solution, the impurity such as phenols can be removed, it is ensured that proposed DNA mass；The method can matched liquor removing workstation, realize it is high throughput automated.
Description
Technical field
The present invention relates to the extracting methods of DNA, specifically, being related to a kind of method that high throughput maize leaf DNA is extracted.
Background technique
It can satisfy the requirement of KASP genotyping, Array using the maize leaf DNA that the methods of CTAB is extracted
Preferable result can be obtained on tape.But time-consuming for this method, and flux is low, and it is toxic organic molten to use a large amount of chloroforms etc.
Liquid is only applicable to the experiments high for DNA quality requirement such as sequencing or genetic chip, and extensive high-throughput leaf DNA is mentioned
It takes such as identified for genes, is sheerly material and parting etc. is marked then to be not suitable for very much on a small quantity.Therefore exploitation is without using toxic organic reagent
The method of rapidly extracting maize leaf DNA is very important.
" a kind of cotton leaf DNA rapid extracting method " (" Cotton Science ", 04 phase in 2016) proposed by Zhang Youchang,
It is understood that cotton leaf DNA can be extracted by adding KCl to carry out boiling water bath using TE buffer, which can satisfy regular-PCR reality
The requirement tested.However, still needing to develop a kind of method that can be carried out high-throughput maize leaf DNA and extract, so that being extracted
DNA not only can satisfy common PCR reaction, but also can satisfy KASP Genotyping and trace P CR system (≤5 μ L) reaction
Requirement.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of high-throughput maize leaf DNA
The method of extraction.
In order to achieve the object of the present invention, technical scheme is as follows:
The present invention provides a kind of method that high throughput maize leaf DNA is extracted, comprising:
Pretreatment fluid is added in maize leaf, is ground；
Pretreatment fluid is sucked out to discard, DNA extracting solution is added；
Water-bath is extracted after mixing, and extracting solution is centrifuged, and takes supernatant, and isopropanol precipitating DNA is added, and is mixed, and centrifugation discards
Supernatant；
Be added 75% alcohol wash precipitating DNA, mix, centrifugation, discard supernatant to get.
Wherein, the pretreatment fluid and the DNA extracting solution contain 0-4%m/v PVP, preferably comprise 2%m/v PVP.
More preferably, the pretreatment fluid and the pre- DNA extracting solution are using following proportion:
| Reagent | Pretreatment fluid | DNA extracting solution |
| PVP40 | 2g | - |
| PVP30 | - | 2g |
| 1M Tris-HCl | 5mL | 10mL |
| 0.5M EDTA | 0.5mL | 1mL |
| 3MKcl | - | 33mL |
| ddH2O | 93.5mL | 56mL |
| Beta -mercaptoethanol | 1mL | - |
| It is total | 100mL | 100mL |
Products therefrom can be dried using 37 DEG C of baking ovens, add water, spare.
Further, when maize leaf sample is a small amount of or when not needing mailing, can directly be extracted fresh blade
(each sample about 30mg)；When maize leaf sample is a large amount of or when needing to post, maize leaf is put into appropriate containers, is set
Drying is refrigerated in freeze drier.
Generally, 96 orifice plates (such as the hole NUNC96 deep-well plates) or ELISA Plate can be used as container, it both can lid silica gel lid
Aluminium foil paper membrane can also be sealed up.
Further, conventional method progress can be used in grinding of the present invention, such as appropriate steel ball is added when grinding (directly
Diameter 6mm), or ground using high-throughput animal vegetable tissue grinder (genogrinder), it usually can be in 1200-1400rpm
Grind 45s-1min (such as 1200rpm grinds 1min).
Preferably, the pretreatment fluid of about 10 times of volumes of sample size is added before the milling, plays and maize leaves are effectively reduced
The effect that piece is oxidized during the grinding process.
Preferably, the pretreatment fluid and the DNA extracting solution contain 0-4%m/v PVP, further preferably 2%
m/v PVP。
Preferably, the additional amount of the DNA extracting solution is suitable with pretreatment fluid.
Preferably, the Extracting temperature that the water-bath is extracted is 90-95 DEG C, further preferably 93 DEG C.
Preferably, the extraction time that the water-bath is extracted is 30-45min, further preferably 30min.
Preferably, in the extraction process that the water-bath is extracted simultaneously or interruption (such as interruption 10min) carries out at mixing
Reason (as used vortex oscillator).
Preferably, 0.6-1 times volume of the dosage of the isopropanol by taking supernatant after water-bath, preferably 0.6 times
Volume.
Preferably, the centrifugation being related in the method for the invention, condition is to be centrifuged 10- in 3600-5300r pm
20min (such as 4000rpm is centrifuged 10min).
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined with each other, obtain specific embodiment party
Formula.
Further, the present invention provides a kind of method that specific high throughput maize leaf DNA is extracted, including walks as follows
It is rapid:
(1) pretreatment of about 10 times of volumes is added in the fresh blade of 30mg or so (or after equivalent blade is freeze-dried)
Liquid is centrifuged after grinding；
(2) supernatant is abandoned, is added and is mixed with the DNA extracting solution of pretreatment fluid equivalent, be placed in water-bath in 90-95 DEG C of water-bath
30-45min, during which concussion mixes, and is centrifuged after the water bath is over；
(3) supernatant is taken, 0.6 times of volume isopropanol is added, mixes precipitating DNA, supernatant is abandoned in centrifugation；
(4) be added volume fraction be 75% ethyl alcohol, mix centrifugation, abandon supernatant to get.
More specifically, in an embodiment of the invention, the method that the high throughput maize leaf DNA is extracted,
Include the following steps:
1. taking about 30mg maize leaf into 96 orifice plate of 2mL, 96 orifice plates are placed in freeze drier and were freeze-dried
Night；
2. taking out 96 orifice plates, 1 6mm steel ball is added in every hole, and every hole adds 400 μ L pretreatment fluids, covers silica gel rapidly
Film is placed in genogrinder, and 1200rpm is ground 45 seconds, and 4000rpm is centrifuged 10min；
3. tearing pellosil, Aspirate supernatant 200-300 μ L is discarded, and the DNA extracting solution of 400 μ L is added in every hole, covers silicon
Glue film mixes well on the oscillator of fine jade whirlpool；
4. 96 orifice plates are placed in water-bath 30-45min in 90-95 DEG C of water-bath, the taking-up of 96 orifice plates is placed in fine jade every 10min
It shakes and mixes on the oscillator of whirlpool, 4000rpm is centrifuged 10min；
5. drawing 200-250 μ L supernatant into 96 new orifice plates, 0.6 times of volume isopropanol is added, mixes precipitating
DNA30min or more；
6. taking out 96 orifice plates, 4000rpm is centrifuged 5min, discards supernatant；
7. 500 μ L, 75% ethyl alcohol is added in every hole, mix, 4000rpm is centrifuged 5min, discards supernatant；Natural air drying or 37 DEG C
It dries 2-3 hours, is dissolved in water spare in baking oven.
The invention also includes application of the above method on KASP genotyping.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
Beneficial effects of the present invention at least that:
The method of the invention carries out sample process without using liquid nitrogen, and without using organic solvents such as chloroforms, relatively
It is safe and non-toxic；The present invention effectively prevents the oxidation of maize leaf during the grinding process, and locating in advance by the way that pretreatment fluid is added
The PVP40 of addition 2% in liquid is managed, while 2% PVP30 being added in DNA extracting solution, PVP can form a kind of insoluble with polyphenol
Complex compound, effectively remove polyphenol, reduce the pollution of phenol in proposed maize leaf DNA, while it also can be effective in conjunction with polysaccharide
Polysaccharide removing, it is ensured that mentioned DNA mass；The method can matched liquor removing workstation, realize it is high throughput automated.
Detailed description of the invention
Fig. 1 is the method flow diagram that high-flux rice leaf DNA of the present invention is extracted.
Fig. 2 is the genotypic results figure in experimental example 1.
Specific embodiment
Below with reference to embodiment the present invention will be further explained explanation.It will be appreciated that following embodiment provides
Merely to playing the purpose of explanation, it is not used to limit the scope of the present invention.Those skilled in the art is not
In the case where spirit of the invention and spirit, the present invention can be carry out various modifications and be replaced.
Experimental method used in following embodiments is conventional method unless otherwise specified, can be according in the art
Document described in technology or conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molec μ Lar Cloning:a Laboratory Manual, 2001), or carried out according to product description.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The present embodiment is used to illustrate the extracting method of maize leaf DNA, and steps are as follows, and (the extraction flow diagram of DNA is shown in
Fig. 1):
1. taking about 30mg maize leaf into 96 orifice plate of 2mL, it is small that 96 orifice plates are placed in freeze drier dry about 12
When；
2. taking out 96 orifice plates from freeze drier, 1 6mm steel ball is added in every hole, and every hole adds 400 μ L pretreatment
Liquid covers rapidly pellosil, is placed in high-throughput animal vegetable tissue grinder (genogrinder), and 1200rpm is ground 45 seconds
Afterwards, 4000rpm is centrifuged 10min；
3. tearing pellosil, Aspirate supernatant 200-300 μ L is discarded, and 400 μ L DNA extracting solutions are added in every hole, covers silica gel
Film mixes well on the oscillator of fine jade whirlpool；
4. 96 orifice plates, which are placed in water-bath 30-45min in 90-95 DEG C of water-bath, (is placed in fine jade for the taking-up of 96 orifice plates every 10min
Shake and mix on the oscillator of whirlpool) after, 4000rpm is centrifuged 10min；
5. drawing 200-250 μ L supernatant into 96 new orifice plates, 0.6 times of volume isopropanol is added, mixes precipitating DNA
30min or more；
6. taking out 96 orifice plates, 4000rpm is centrifuged 5min, discards supernatant；
7. 500 μ L, 75% ethyl alcohol is added in every hole, mix, 4000rpm is centrifuged 5min, discards supernatant；
8. being dried 2-3 hours in natural air drying or 37 DEG C of baking ovens, it is dissolved in water spare.
In abovementioned steps, the formula of used pretreatment fluid and DNA extracting solution is as follows.
| Reagent | Pretreatment fluid | DNA extracting solution |
| PVP40 | 2g | - |
| PVP30 | - | 2g |
| 1M Tris-HCl | 5mL | 10mL |
| 0.5M EDTA | 0.5mL | 1mL |
| 3MKcl | - | 33mL |
| ddH2O | 93.5mL | 56mL |
| Beta -mercaptoethanol | 1mL | - |
| It is total | 100mL | 100mL。 |
Experimental example 1
Using 93 parts of maize leaf materials (existing CTAB method genotype), method described in Application Example 1 extracts gene
Group DNA, carries out parting on KASP genotyping system.Reaction kit is the IntelliQube full-automation PCR/qPCR of LGC
Establishing, amplification and analysis system.
Reaction system are as follows: the Primer of the DNA of 0.8 μ L, 2 × KASP Master mix (LGC) of 0.8 μ L, 0.022 μ L
mix(ThermoFisher)。
Primer mix used in the present invention is following 3 groups:
SNP559:
Forward primer 1:GAAGGTGACCAAGTTCATGCTTGCTGCTGACAGGAGGAAGAT；
Forward primer 2:GAAGGTCGGAGTCAACGGATTTGCTGCTGACAGGAGGAAGAC；
Reverse primer: ATGAACCAGTTGTTGATCTGCTTC；
Response procedures are as follows:
Experimental result is as follows:
This method and CTAB method compare, and as a result see Fig. 2.Gene point is carried out using the DNA that 1 the method for embodiment is extracted
Type, sub-clustering is obvious, and each sub-clustering result is also concentrated, and NTC is located at origin.According to experimental result it is found that the method for the invention can be with
Meet KASP and detect 1.6 μ L reaction systems, and finds out the result basic one of this method Yu CTAB method from final genotyping result figure
It causes.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Yuanlongping Agricultural Hi-Tech Co., Ltd.
<120>a kind of method that high throughput maize leaf DNA is extracted
<141> 2018-10-16
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaaggtgacc aagttcatgc ttgctgctga caggaggaag at 42
<210> 2
<211> 42
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gaaggtcgga gtcaacggat ttgctgctga caggaggaag ac 42
<210> 3
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
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Claims (10)
1. a kind of method that high throughput maize leaf DNA is extracted characterized by comprising
Pretreatment fluid is added in maize leaf, is ground；
Pretreatment fluid is sucked out to discard, DNA extracting solution is added；
Water-bath is extracted after mixing, and extracting solution is centrifuged, and takes supernatant, and isopropanol precipitating DNA is added, and is mixed, and centrifugation discards
Clearly；
Be added alcohol wash precipitating DNA, mix, centrifugation, discard supernatant to get；
Wherein, the pretreatment fluid and the DNA extracting solution contain 0-4%m/v PVP.
2. the method according to claim 1, wherein the pretreatment fluid and the DNA extracting solution contain 2%m/
v PVP。
3. according to the method described in claim 2, it is characterized in that, the pretreatment fluid and the DNA extracting solution are using as follows
Proportion:
4. described in any item methods according to claim 1~3, which is characterized in that the Extracting temperature that the water-bath is extracted is 90-
95 DEG C, further preferably 93 DEG C.
5. according to the method described in claim 4, it is characterized in that, the water-bath extract extraction time be 30-45min, into
One step is preferably 30min.
6. method according to claim 4 or 5, which is characterized in that in the extraction process that the water-bath is extracted simultaneously or
It is disconnected to carry out mixing processing.
7. according to the method described in claim 6, it is characterized in that, the dosage of the isopropanol is by taking supernatant after water-bath
0.6-1 times of volume.
8. described in any item methods according to claim 1~7, which is characterized in that the condition of the centrifugation is 3600-
5300rpm is centrifuged 10-20min.
9. a kind of method that specific high throughput maize leaf DNA is extracted, which comprises the steps of:
(1) maize leaf after fresh corn blade or freeze-drying is added to the pretreatment fluid of 10 times of volumes, is centrifuged after grinding；
(2) supernatant is abandoned, is added and is mixed with the DNA extracting solution of pretreatment fluid equivalent, be placed in water-bath 30- in 90-95 DEG C of water-bath
45min, during which concussion mixes, and is centrifuged after the water bath is over；
(3) supernatant is taken, 0.6 times of volume isopropanol is added, mixes precipitating DNA, supernatant is abandoned in centrifugation；
(4) be added volume fraction be 75% ethyl alcohol, mix centrifugation, abandon supernatant to get；
Wherein, the pretreatment fluid and the DNA extracting solution contain 2%m/v PVP.
10. according to the method described in claim 9, it is characterized in that, the pretreatment fluid and the DNA extracting solution are using as follows
Proportion:
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| CN201811251487.5A CN109371009A (en) | 2018-10-25 | 2018-10-25 | A kind of method that high throughput maize leaf DNA is extracted |
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Cited By (2)
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| CN111961662A (en) * | 2020-08-31 | 2020-11-20 | 江西农业大学 | DNA extracting solution and method for extracting sesame leaf genome DNA |
| CN112779247A (en) * | 2021-01-19 | 2021-05-11 | 袁隆平农业高科技股份有限公司 | Plant tissue genome DNA extraction kit and high-throughput extraction method |
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| CN101575597A (en) * | 2009-05-21 | 2009-11-11 | 中国农业大学 | Kit for quickly extracting plant genome and applications thereof |
| CN102296063A (en) * | 2011-08-30 | 2011-12-28 | 华南农业大学 | Extraction solution for extracting DNA of plant and extraction method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111961662A (en) * | 2020-08-31 | 2020-11-20 | 江西农业大学 | DNA extracting solution and method for extracting sesame leaf genome DNA |
| CN111961662B (en) * | 2020-08-31 | 2021-11-02 | 江西农业大学 | DNA extracting solution and method for extracting sesame leaf genome DNA |
| CN112779247A (en) * | 2021-01-19 | 2021-05-11 | 袁隆平农业高科技股份有限公司 | Plant tissue genome DNA extraction kit and high-throughput extraction method |
| CN112779247B (en) * | 2021-01-19 | 2022-07-08 | 袁隆平农业高科技股份有限公司 | Plant tissue genome DNA extraction kit and high-throughput extraction method |
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